Bioengineered Salivary Gland Microtissues─A Review of 3D Cellular Models and their Applications

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Bioengineered Salivary Gland Microtissues�A Review of 3D Cellular
Models and their Applications
Sangeeth Pillai, Jose G. Munguia-Lopez, and Simon D. Tran*
Cite This: https://doi.org/10.1021/acsabm.4c00028
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ABSTRACT: Salivary glands (SGs) play a vital role in maintaining oral health through the production and release of saliva. Injury
to SGs can lead to gland hypofunction and a decrease in saliva secretion manifesting as xerostomia. While symptomatic treatments
for xerostomia exist, eective permanent solutions are still lacking, emphasizing the need for innovative approaches. Significant
progress has been made in the field of three-dimensional (3D) SG bioengineering for applications in gland regeneration. This has
been achieved through a major focus on cell culture techniques, including soluble cues and biomaterial components of the 3D niche.
Cells derived from both adult and embryonic SGs have highlighted key in vitro characteristics of SG 3D models. While still in its first
decade of exploration, SG spheroids and organoids have so far served as crucial tools to study SG pathophysiology. This review,
based on a literature search over the past decade, covers the importance of SG cell types in the realm of their isolation, sourcing, and
culture conditions that modulate the 3D microenvironment. We discuss dierent biomaterials employed for SG culture and the
current advances made in bioengineering SG models using them. The success of these 3D cellular models are further evaluated in the
context of their applications in organ transplantation and in vitro disease modeling.
KEYWORDS: Salivary glands, Three-dimensional culture, Bioengineering, Spheroids, Organoids, Extra cellular matrix, Transplantation,
Disease modeling
■INTRODUCTION
Salivary glands (SGs) are exocrine structures that are tasked
with the production and release of saliva. In humans, these
glands fall into two primary categories: the major paired
glands�namely, the parotid (PA), submandibular (SMG), and
sublingual (SLG) glands and the numerous minor salivary
glands (600−1000) dispersed throughout the upper aero-
digestive tract, encompassing regions such as the lips, palate,
cheeks, and tongue.
1
The SGs are structured as intricately
branched formations, comprising various cell types that
collaborate harmoniously to respond to physiological signals
and facilitate the secretion of saliva.
2
Damage to these distinct
salivary cell types can impact both the quality and quantity of
saliva produced, potentially leading to conditions such as dry
mouth or xerostomia.
3,4
Radiation-induced SG hypofunction in
individuals undergoing treatment for head and neck cancers,
along with development of Sjogren’s Syndrome�an auto-
immune disease�stand as the primary contributors to
xerostomia.
5−7
Currently, no permanent solution exists to
eectively treat severe xerostomia aecting this patient
population.
8
Most treatments are symptomatic, involving the
use of secretagogues and artificial saliva and several others
struck in the clinical pipeline.
9−11
Presently, only three
medications have received approval for a lasting remedy for
xerostomia, the rest solely focusing on symptom alleviation. Of
these, pilocarpine and cevimeline are only eective in cases
Received: January 8, 2024
Revised: March 30, 2024
Accepted: April 2, 2024
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where patients have remaining acinar/progenitor population
that are capable of being stimulated.
12
Amifostine, on the other
hand, has shown significant side eects when administered
intravenously, underscoring the need for further research to
develop eective treatments for xerostomia.
6,13
Eorts to address salivary gland hypofunction and
xerostomia have led to the emergence of three interdisciplinary
research focus areas: bioengineering of an artificial SG
gland,
14−18
use of SG and non-SG stem cell therapy,
19−22
and delivery of gene therapy.
23−27
A substantial body of
research has concentrated on the first two aspects, aiming to
restore and regenerate the lost gland function. Various research
groups have undertaken the challenge of optimizing the SG cell
culture, facing inherent diculties due to the slow proliferation
potential and dynamic phenotypic characteristics in vitro.
28−31
To address challenges associated with two-dimensional (2D)
culture systems, researchers have turned to the development
and utilization of innovative biomaterials to mimic and serve as
the native extracellular matrix (ECM).
18,28,32
A variety of
natural, synthetic, and composite biomaterials have been
employed to support SG cell growth in three-dimensional
(3D) culture for extended periods and for use as trans-
plantation scaolds.
14,33,34
These advancements have also
enhanced our understanding of the various SG cell types,
including the diversity of stem/progenitor cell populations,
which are crucial as treatment targets or for use in stem cell
therapy.
35−37
Furthermore, eorts have been directed toward
comprehending how ECM remodelling and the physicochem-
ical properties of biomaterials influence the SG phenotype and
secretory functions, which are paramount for successful clinical
applications.
34,38,39
Clinical trials, on the other hand, have
centered on delivering stem cells and their derivatives from
dierent sources to alleviate xerostomia. In recent times, a
handful of human clinical trials have demonstrated the
promising potential of autologous mesenchymal stem cells
(MSCs) in the treatment of radiation-injured salivary
glands.
40−42
However, these studies are ongoing and are
constrained by several limitations until they are made clinically
available. Moreover, there is a need for extended, long-term
investigations to validate the safety and ecacy of these cell-
based treatments in humans. While existing research has
provided valuable insights into the fundamentals of SG
regeneration, a common consensus suggests that a synergistic
combination of approaches is needed to address the diverse
challenges involved.
In this review, our focus is on the strides made in the field of
3D SG bioengineering over the past decade. To gather relevant
information, we conducted a comprehensive literature review
using the MEDLINE and PubMed databases. The search
utilized the term “salivary gland” in conjunction with the
Boolean operator “AND,” combining it with search terms such
as “bioengineering,” “biomaterials,” “3D culture,” “spheroids,”
and “organoids.” The search results were limited to studies
published between 2013 and 2023. From MEDLINE, we
identified 144 studies, and from PubMed, 186 studies, totaling
330 studies. After screening for duplicates and excluding
reviews and nonrelevant studies, an evaluation was conducted
on 77 studies, forming the basis for the compilation of this
narrative manuscript. Based on our search results, we first delve
into the importance of dierentiated and progenitor SG cell
Figure 1. A schematic workflow illustrating commonly employed strategies for isolation, and culture of SG cells in the bioengineering of 3D
models. A) Adult major SGs are enzymatically digested to obtain single cells or SG cell clusters which are either grown as adherent cultures or as
cell clusters in suspension or low attachment plates. Single cells containing both dierentiated and progenitor cell populations can then be sorted to
obtain a specific population or used directly and are capable of forming secondary spheres or organoids. These organoids/spheres can be digested
into single cells (that can be used in stem cell transplantation), with a potential to self-renew and form newer organoids/spheres. The secondary
spheres can be further cultured with specific growth factors and soluble cues with or without scaolds/ECM to form dierentiated ASC-derived SG
organoids. B) Developing mouse embryonic SGs (mainly SMGs) are isolated at the E12-E14 stage. The embryonic glands either are enzymatically
digested to obtain single cells or are first dissected to separate the epithelium and the mesenchyme. Single cells (epithelial and mesenchymal) can
then be obtained by digesting the dierent gland compartments for downstream applications. Alternatively, sections of the epithelial and
mesenchymal components can be isolated and reconstituted with the help of supporting matrices to form the SG organ germ and transplanted into
in vivo models. Epithelial and mesenchymal cells can be cocultured to study SG organogenesis (e.g., branching morphogenesis) or used as cell
populations for PSC-based SG organoid formation. Dierentiated and functional ex vivo cultured embryonic gland-derived SG organoids can be
used downstream for transplantation or for use in in vitro testing.
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types, highlighting their sourcing, isolation, and culture
practices for 3D applications. Subsequently, we explore the
advancements in biomaterial strategies, combined with the use
of soluble cues that have facilitated the successful culture of
these SG cells using dierent 3D platforms. Lastly, we discuss
the current status of bioengineered SG cell models, examining
their potential applications in organ transplantation and disease
modeling.
1. UNDERSTANDING THE SG CELL
NICHE�SOURCING AND ISOLATION STRATEGIES
FOR 3D CULTURE
Cells constitute a cornerstone in the realm of bioengineering
within organ systems, playing a crucial role in the translational
application of bioengineered tissues. Adult SGs consist of three
major epithelial cell types: the acinar, ductal, and myoepithelial
cells. Acinar cells serve as the primary secretory cells
responsible for the production and secretion of saliva.
Depending on the composition and type of secretory proteins,
acinar cells can be classified as serous or mucous cells.
43
The
ductal architecture of SGs encompasses striated, intercalated,
and excretory ducts, collectively regulating saliva composition
and facilitating its transport to the oral cavity. Lastly,
myoepithelial cells assume a pivotal role by furnishing physical
and neural cues that stimulate saliva secretion from acinar cells
in response to stimuli. Comprehending and harnessing the
function of these distinct cell types are imperative for the
ecient bioengineering of functional and clinically pertinent
SG models. Cell lines have commonly served as convenient cell
sources and biological models for various applications
including tissue bioengineering. However, the heterogeneity
and dynamic nature of SG cell populations, crucial for
replicating phenotypic and functional aspects of native glands,
impose limitations on the use of a single cell line for studying
their physiology. Additionally, while nonhuman SG cell lines
exist, the dierences in the physiology of these species limit
their use for translational studies. The sole documented and
normal SG-derived cell line was established in the early 1990s
and has had limited applications over the years.
44
Recently,
mice SMG-derived immortalized cell lines have been
established and characterized, showing the ability to self-
organize into 3D spheroids.
45,46
A detailed review on the
dierent cell lines used for SG research can be found here.
47
Eorts to bioengineer an artificial SG using primary cells
began nearly two decades ago, with an aim to establish
polarized epithelial cell cultures using transwell plates.
48
Since
then, numerous methods and strategies have been elucidated
to isolate specific SG cell populations from adult or embryonic
tissues, both for in vitro culture systems and for utilization in in
vivo transplantation studies. Typically, cell isolation strategies
from adult SGs focus on obtaining epithelial cells or SG adult
stem/progenitor cells (ASCs) and pluripotent stem cells
(PSCs) such as embryonic stem cells (ESCs) or induced
pluripotent stem cells (iPSC) from developing mice SGs.
These cells are further subjected to functional and molecular
characterization as 2D monolayer cultures, as on-top cultures,
as self-assembled spheres or by using ECM mimetics like
Matrigel, synthetic hydrogels or by the use of bioprinting
(fabrication) techniques to form 3D spheroids or organoids.
Figure 1 shows the strategies used in the sourcing, isolation,
and culture of SG cells in the context of the bioengineering of
3D SG models. These models are downstream useful to study
SG disease mechanisms and employed toward organ or stem
cell transplantation.
1.1. Isolation and Culture of SG Epithelial Cells.
Primary SG epithelial cells are characterized by the presence of
key epithelial junctional proteins like E-cadherins (E-cad)
forming adherence junctions (AJs), zona-occludins-1 (ZO-1)
forming tight junction proteins (TJs), and dierent claudins
and occludins depending on the gland type.
48,49
Shin et al.
isolated and cultured human PA gland epithelial cells (hPEC)
on dierent surfaces including plastic, Matrigel, polyethylene
glycol (PEG), and hydrogel micropatterned-polycaprolactone
(PCL) nanofibrous microwells.
50
The hPEC aggregated to
form 3D acinar-like spheroids on Matrigel, PCL, and PCL
microwells and expressed key epithelial markers like ZO-1,
occludins, E-cad, and acinar markers like aquaporins-5 (AQP5)
and α-amylase. The authors concluded that PCL microwells
provide a better environment, compared to Matrigel for SG
epithelial cells to reorganize into acinar like spheroids, making
them suitable for bioengineering of ex vivo SG models.
50
Beucler et al. outlined a detailed protocol for the isolation of
human salivary gland (SG) epithelial cells intended for
cultivation as salispheres or adherent monolayer cultures.
The protocol yields a heterogeneous population primarily
comprising epithelial cells that can be successfully propagated
for 5−8 passages, or 15−20 population doublings, before
undergoing senescence.
51
One of the major challenges in
working toward SG regeneration is the low availability of
human samples for testing. To overcome this, eorts have been
directed toward the cultivation of dierentiated secretory
epithelial cells sourced from porcine SMG and PA glands.
These cells express essential epithelial lineage markers,
allowing for valuable insights and potential applications
translating to culturing human SG cells.
52
We recently
published a comprehensive protocol for isolating and culturing
single cells from human and mice SG tissues under serum-free
conditions.
29
Acinar or ductal SG epithelial cells can be
obtained either from small tissue explants (∼1−2 mm in size)
or through digestion using Liberase enzymes and a
gentleMACS dissociator, rendering them suitable for down-
stream applications.
29
Nonetheless, maintaining primary SG cells in a single-cell
state is commonly quite challenging due to issues such as slow
cell proliferation, a high rate of dedierentiation, and
alterations associated with epithelial-to-mesenchymal transi-
tions (EMT) in glandular organs.
53−55
This has led to the
development of alternative culture practices to maintain both
progenitor and dierentiated epithelial populations isolated
from adult SG tissues. Leigh et al. proposed culturing mice SG
tissue derived cells as clusters in growth factor reduced (GFR)-
Matrigel to maintain epithelial cell characteristics in addition to
sustaining agonist-induced secretory (amylase) function.
56
Our
research group went on to develop and refine a serum- and
matrix-free culture system designed to sustain dierentiated
epithelial cells derived from human SG tissue. This innovative
approach involved maintaining these cells as clusters, termed
salivary functional units (SFUs), within a scalable suspension
system.
57
The partially dissociated clusters were found to
contain gland-specific ECM, supporting cell growth and
maintaining acinar and ductal epithelial markers for more
than a week in culture.
57
This method provides an alternative
strategy for preserving SG cell characteristics, including cell
polarization, and in long-term matrix-supported cultures for
organoid formation. More recently, Song et al. proposed
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encapsulation of primary SG acinar cell clusters and
intercalated ducts (AIDUCs) in 3D hydrogels to form SG
microtissues.
58
The authors reported that the cell clusters
maintained key SG-specific markers like NKCC1, PIP, and
AQP5 in 3D for 14 days in culture and responded to
stimulation by purinergic agonists.
1.2. Isolation and Culture of SG-Derived Adult Stem
Cells. The ectoendodermal origin of SG tissue-resident stem
cells or ASCs position them as optimal candidates for
implementation in the bioengineering of dysfunctional glands.
In an initial report, Nanduri et al. detailed the purification and
isolation of fully functional SG stem cells from mice SG
tissues.
59
Mice SGs were enzymatically digested, leading to the
growth of cell clusters or salispheres. Subsequent dissociation
into single cells and cell sorting facilitated the formation of
secondary spheres. These secondary spheres were then
dierentiated to generate SG organoids or dissociated into
single cells, allowing for self-renewal and sphere formation for
over 12 passages (Figure 1A). The authors observed that single
cells derived from salispheres exhibited stem/progenitor
properties. Following fluorescence-activated cell sorting
(FACS), CD24hi/CD29hi cell clusters demonstrated the
highest self-renewal potential, suggesting their potential as
ideal candidates for stem cell therapy in SG hypofunction.
59
In
a subsequent study, the research group reported that the long-
term expansion potential of SGSCs (salivary gland stem cells)
is driven by WNT signaling. They specifically highlighted the
existence of SGSCs in salivary gland ductal regions by isolating
rare EPCAM+ (epithelial cell adhesion-molecule-positive) SG
ductal cells. These cells were found to harbor the potential for
activation, sustaining long-term in vitro expansion, self-
renewal, and the formation of organoids.
60
Recently, Lee et
al. achieved successful isolation of salispheres from all major
salivary glands in mice.
61
The group reported that these
primary spheres exhibited distinctive cellular characteristics
specific to the type of gland. Furthermore, they transplanted
and tracked the presence of eGFP-labeled salispheres derived
from all major glands in dierent parts (acinar and ductal) of
the SMG of irradiated mice, indicating the potential use of
salispheres from dierent gland types or regions for trans-
plantation purposes.
61
Building on the success observed with mice SGSCs, Pringle
et al. investigated the self-renewal and dierentiation potential
of human SGSCs.
36
The authors documented that ASCs
obtained from human SG tissues, when cultured as salispheres,
demonstrated the capacity for self-renewal and dierentiation
into functional SG organoids from single cells. Moreover, when
transplanted into an irradiated mouse model, these stem cells,
especially c-kit+ cells, exhibited the highest self-renewal
potential and eectively rescued SG hypofunction. These
findings led the authors to conclude that human SGSCs,
particularly c-kit+ stem cells, present significant therapeutic
potential for restoring organ function postirradiation injury.
36
ASCs isolated from human SG has been lately investigated on
their potential to dierentiate into SG organoids based on
microenvironmental and soluble cues.
62,63
Recently, Yoon et
al. devised a culture protocol for generating SG organoids from
both mice and humans in a Matrigel culture system.
64
These
ASC-derived organoids demonstrated the potential for long-
term in vitro expansion, stable maintenance, and ability to
respond to functional stimulation. Single-cell RNA sequencing
confirmed the expression of key SG markers, indicating the
representation of essential cell heterogeneity similar to the
native gland tissues.
64
While SGSC cultures exhibited
significant potential for self-renewal and organoid formation
in Matrigel, concerns about the clinical translation of animal-
derived matrices, especially for stem cell or organoid
transplantation, prompted researchers to explore alternatives.
65
Srinivasan et al. developed a hyaluronic-acid (HA) based 3D
hydrogel system to replace the suspension-based, salisphere
culture of SGSCs.
63
The authors reported that isolated SGSCs
exhibited high expression of progenitor markers such as K5,
K14, MYC, ETV4, and ETV5. Additionally, these SGSCs
demonstrated the ability to form primary and secondary
spheres in a suspension. When cultured in 3D modular
hyaluronate hydrogels modified with basement membrane-
derived peptides, the SGSCs exhibited enhanced viability,
stemness, and expression of progenitor markers.
63
Other
eorts to replace animal-based 3D matrices for culturing and
expanding SGSCs include the use of magnetic bio
assembly
66−68
or microwell-based
69,70
platforms to isolate
and culture SGSCs and are discussed in the next section.
1.3. Isolation and Culture of Embryonic SG Cells.
Functional adult SGs develop from the SG organ germ, which
is formed through simultaneous epithelial−mesenchymal
interactions. This intricate process, coupled with surrounding
ECM niche signaling, initiates branching morphogenesis, cell
dierentiation, and maturation, ultimately giving rise to fully
functional SGs.
71
To date, developing mouse SMGs have
proven to be an ideal system for studying the complex
organogenesis processes of SGs (Figure 1B). They show
excellent potential to grow in ex vivo conditions and display
mechanisms similar to in vivo development, making them
valuable models for investigating the complex, sequential
processes involved in SG organogenesis.
72
To leverage the potential of the SG organ germ, Ogawa and
Tsuji developed a protocol for reconstituting and bioengineer-
ing the epithelial and mesenchymal components of developing
mice SMGs (E13-E14) for organ transplantation.
73
This
method was noted for its advantages, not only in replacing
damaged SGs but also in comprehending ex vivo the SG-
specific cell kinetics and regulation of crucial genes that can be
targeted for future therapies.
17
Ono and colleagues introduced
an approach that combines the use of iPSCs with embryonic
SMG cells (iSG) to address challenges associated with iPSC
tumorigenesis and enhance the properties of cultured
embryonic SMG cells.
74
They reported that E13.5 SMG
derived cells grown in the presence of iPSCs developed more
epithelial structures than SMGs alone, suggesting the potential
of iPSCs in accelerating SG dierentiation. It was only recently
that iPSC-derived SG organoids were developed for use as in
vitro models to study viral replication in SG organoids and for
in vivo transplantation.
75
In an eort to gain deeper insights
into the niche requirements of embryonic SMGs, Hosseini et
al. conducted experiments where E16 SMG cells were cultured
under various conditions, including co-culture with fibroblast
cells, mesenchymal cell derived condition media, or embryonic
SG mesenchyme.
76
Their findings indicate that E16 epithelial
cells cultured with their mesenchymal counterparts in the
presence of FGF2 signaling exhibited robust expression of
progenitor markers, including Kit proteins, and acinar marker
AQP5 in SG organoids.
76
The group further reported a
detailed protocol describing the steps to isolate, separately,
epithelial and mesenchymal cells from embryonic SG to
develop and manipulate organoids.
77
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More recently, Moskwa et al., utilizing single-cell RNA
sequencing, reported that PDGFRα+ cells, a subset of stromal
cells present in E16 SMGs, have the potential to promote
proacinar cell dierentiation in SG organoids.
78
They further
found that this potential is regulated by FGF2 signaling, which
plays a crucial role in activating BMP genes in these stromal
cell populations. An alternative source to study SG organo-
genesis was reported by Zhang et al., using PSCs.
79
The
authors demonstrated that a stepwise treatment involving
retinoic acid and FGF10 promoted the dierentiation of PSCs
into salivary gland placodes and successfully recapitulated the
early morphogenetic events associated with salivary gland
development.
79
These placodes were successfully used for in
vivo transplantation, as discussed later in this review. Just
recently, the group further reported a detailed protocol for
dierentiating these human PSCs into SG epithelial progen-
itors expressing key makers including SOX9, K5, and K19.
80
RNA sequencing analysis of the dierentiated epithelial
progenitors showed shared transcriptomic profiles with fetal
SMGs.
SGSCs play a crucial role in stem cell therapy for alleviating
salivary gland hypofunction. Their inherent ability to self-
renew not only facilitates stem cell therapy but also contributes
to the creation of functional 3D cellular models. These models
serve as valuable tools for studying salivary gland pathophysi-
ology ex vivo and hold potential for organ replacement
therapies. Detailed descriptions on the distinct types SGSCs
found in adult and embryonic SGs and their applications in SG
regeneration can be found in previous reviews.
20,81
2. THE BIOMATERIALS PERSPECTIVE�ADDING A
THIRD DIMENSION TO SG CELL CULTURE
Naturally, mammalian cells are in constant communication
with other cells and with their ECM in a well-organized and
complex 3D configuration. Dierent cell types require specific
ECM conditions, such as the viscoelastic and degradation
properties of the matrix, to maintain homeostasis and function
using biological feedback loops.
82
Three-dimensional cell
culture in ECM-like materials is used to create spheroids and
organoids in vitro to understand and elucidate the biological
mechanisms of cells in health and disease. Since the inception
of the concept of bioengineering an artificial SG for use in
gland regeneration, researchers have focused on developing
and optimizing ECM mimetics for culturing SG cells. Both
natural and synthetic ECM substitutes have been used
extensively for SG tissue engineering applications. Table 1
outlines a detailed description of the dierent types of natural
and synthetic biomaterials employed for SG tissue engineering
over the past decade with a focus on cell source, soluble cues,
and the 3D culture system employed.
Both scaold-based
50,94−96
and scaold-free, or microwell,
magnetic levitation-based self-organization strategies
66,97,98
have been used in the formation of 3D SG models. Functional
SG spheroid and organoid models have been successfully
established using Matrigel, the most utilized ECM matrix for
3D culture.
59,62,64
However, concerns regarding the use of
animal-derived matrices have forced exploration of other
natural and synthetic alternatives for 3D cell culture.
65
In the
realm of SG spheroids and organoids, HA hydrogels have
shown promising results in the formation of SG 3D cell
models.
99
Alternatively, focus on basement membrane (BM)
components such as laminin,
85
fibronectin,
100
and scaolds
made of perlecan/heparan sulfate proteoglycans
101
have shown
great promise in self-assembly of SG cells into acinar-like SG
spheroids. Recently, laminin peptides in combination with
fibrin hydrogels have also shown promising results in
establishing polarized and lumen-forming SG 3D micro-
tissues.
85,102
2.1. Natural Derivatives Used in Salivary Gland 3D
Culture. As stated, naturally derived biomaterials oer
exceptional biocompatibility toward replicating cell- and
tissue-specific characteristics in vitro; however, they often
remain restricted due to poor mechanical tunability. Recent
investigation has showcased the potential of natural bio-
materials like alginates for use in SG tissue engineering. The
simple ionic cross-linking mechanism provides a structural
tunability to the 3D hydrogels which partially improves
mechanical characteristics of the scaolds for 3D cell culture.
Jorgensen et al. utilized alginate microtubes to develop an
epithelial and mesenchymal cellular coculture system to study
the SG epithelial−mesenchymal interactions which dictate SG
organogenesis.
94
Further, addition of peptide binding motifs
(for example: RGDs) oer cell adhesion sites necessary to
replicate cell−ECM interactions promoting 3D cell reorganiza-
tion and branching morphogenesis, which are key toward 3D
SG models.
89
With a focus to replace the use of Matrigel, our
lab optimized a novel egg-white-alginate-based hydrogel which
has shown the potential to reorganize SG cell lines into 3D
microtissues using on-top cultures.
103
More recently, we
evaluated the potential of other alginate-based hydrogels in
promoting SG cell proliferation, reorganization, and expansion
in a 3D tunable system (unpublished work). Strategic use of
bio fabrication methods, including 3D bioprinting techniques,
can further improve the utilization of the full spectrum of
properties oered by natural biomaterials, especially composite
hydrogels for use in SG bioengineering applications.
To obtain the highest form of biomimicry, researchers have
focused on the use of natural organ-derived matrices in SG cell
culture applications. Our lab tested the potential of residual
connective tissue fibers obtained after enzymatic digestions of
human SMG for use as native ECM for cell growth.
104
Morphometric analysis revealed that the connective tissue
fibers showed native gland-like alignment using high-resolution
electron microscopy and the presence of key BM proteins like
col I, III, and IV histologically. Further, recycled human SMG-
derived connective tissue digests supported attachment and
proliferation of epithelial and fibroblast cells with high viability
for ∼1 week in culture.
104
This strategy also pertains to the use
of decellularized ECM (dECM), which involves sequential
removal of cellular components of an organ and then
processing the native ECM to encapsulate stem/progenitor
cells for 3D organ modeling. dECM have so far been obtained
from rat and porcine SGs and have successfully been used to
promote SG cell adhesion, expansion, and expression of key
secretory and functional proteins.
90,91
However, limited
success has been reported so far in terms of long-term culture
and functionality of these 3D SG models, and their use in
downstream applications is yet to be investigated.
2.2. Synthetic Derivatives Used in Salivary Gland 3D
Culture. Commonly used synthetic ECM mimetics for SG
tissue engineering applications include the use of PEG,
105
PLGA (Poly(lactic-co-glycolic acid)),
96
or use of hybrid
modifications such as MMP-degradable PEG
106
or PEG-
coated PCL scaolds.
50
They bypass the structural limitations
of natural ECM derivatives and allow modulation of
physicochemical and mechanical properties, which dictate
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Table 1. Biomaterials, Cells, and Culture Conditions Used for 3D Salivary Gland Modelling
Biomaterial Cells, 3D system, and culture conditions Key Findings ref
Natural
Matrigel - Cell-laden hydrogel thermogelled at 37 °C. - Single cells self-assemble as spheroids within 7 days. 83
- SG cells from transgenic c-Kit-CreERT2±mice. - Limited proliferative potential for c-Kit+ cells
- Salisphere growing media.
a
- K14+ cells as the major source of salispheres.
- 3D culture maintained for up to 7−8 days. - c-Kit cells are unreliable SG stem cell marker.
- Cell-laden hydrogel thermogelled at 37 °C. - 3D cultured EpCAM cells respond strongly to Wnt signals stimulating self-renewal and long-term
expansion of SG organoids.
60
- Mice SG cells
- Variable medium composition. - Wnt antagonist-pretreated cells transplanted to irradiated mice restore saliva secretion and increase
the number of functional acini in vivo.
- 3D culture maintained for 7−10 days followed by spheroid isolation, single cell dispersion,
and reculture (∼25 times).
- Cell-laden hydrogel thermogelled at 37 °C. - Mice and human SG organoids express gland-specific genes and proteins of acinar, myoepithelial,
and duct cells.
64
- Human and mice adult SG cells.
- Different media formulations for growing and differentiating organoids in the 3D gels. - Organoids respond to neurotransmitter stimulation.
- Organoid subculturing performed every week or biweekly for mouse and human cultures,
respectively.
- Human SMG organoids isolated basal or luminal cells retaining their native characteristics.
- Mice and human 3D organoids cultured for 8 and 4 months, respectively.
Matrigel + Collagen - Cell-laden hydrogel thermogelled at 37 °C. - Establishment of mSG-PAC1 and mSG-DUC1 cell lines. 84
- Murine immortalized submandibular gland epithelial cells (ductal (mSG-DUC1) and pro-
acinar (mSG-PAC1)).
- mSG-DUC1 expresses ductal markers and form lumenized spheroids.
- DMEM: F12.
b
- mSG-PAC1 expresses pro-acinar markers.
- 3D culture maintained for up to 7 days. - FGF2 upregulates aquaporin-5 and the expression of key differentiation markers in mSG-PAC1.
- Cell-laden hydrogel thermogelled at 37 °C. - 3D cultured cells self-renew and differentiate into multilineage organoids. 36
- Human nonmalignant submandibular SG tissue. - In vivo functionality, long-term engraftment, and functional restoration in a xenotransplantation
model.
- DMEM: F12.
c
- Transplanted human salisphere-derived cells restore saliva production in irradiated mice SGs.
- 3D culture maintained for up to 3 weeks.
Matrigel, Laminin-111, or
composite
- Cell-laden hydrogel thermogelled at 37 °C. -Laminin-enriched basement membrane extract or laminin-111, together with exogenous FGF2,
promotes morphogenesis to form salivary epithelial organoids with robust expression of AQP5 in
terminal buds.
76
- Murine epithelial clusters and cocultured with mesenchymal cells. -Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells in coculture reduced
AQP5 levels in organoids even in the presence of FGF2.
- DMEM: F12.
d
-Basement membrane proteins and mesenchyme cell function is crucial for optimal SG function.
- Multiple medium/cell/gel combinations.
- 3D culture maintained for up to 14 days.
Laminin-111 peptides conjugated
Fibrinogen (L-FH)
- Cells seed on coated-peptide-conjugated fibrinogen plates. Fibrinogen gel was polymerized
by incubation at 37 °C with bovine thrombin.
- YIGSR-Fibrin improved morphology and lumen formation in spheroids. 85
- Rat Par-C10 cell line - YIGSR-A99-Fibrin promotes the formation of spheroids and increases the attachment and number
of 3D structures.
- DMEM: F12.
e
- 3D culture maintained for up to 3 days.
- Cells seed on coated-peptide-conjugated fibrinogen plates. Fibrinogen gel was polymerized
by incubation at 37 °C with bovine thrombin.
- L-FH promotes Par-C10 reorganization into lumen forming acinar-like clusters, cell secretory
function and polarization.
86
- Rat Par-C10 cell line; murine C57BL/6J strain.
- DMEM: F12.
e
- L-FH applied to wounded mice improved the expression of the acinar differentiation markers and
saliva secretion.
- 3D culture maintained for up to 10 days.
L-FH and Matrigel - Par-C10 seeded on peptide-conjugated fibrinogen plates. Fibrinogen gel was polymerized
by incubation at 37 °C with bovine thrombin. hHF-MSC mixed with Matrigel and gelled
at 37 °C.
- FGF-7 is responsible for branching morphogenesis. FGF-10 increases proliferation without
promoting migration branching.
87
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Table 1. continued
Biomaterial Cells, 3D system, and culture conditions Key Findings ref
- Coculture of rat parotid Par-C10 cells with hair follicle-derived mesenchymal stem cells
(hHF- MSC).
- DMEM: F12.
e
Addition of FGF-7, FGF-10, or pFGF-10 to L-FH hydrogel. - Control of length and cellularity of branches by modifying the mode of growth factor presentation
and delivery.
- 3D culture maintained for up to 6 days.
RGDPS-functionalized Hyaluronic
acid (HA)
- Cells embedded into precursor before gelation (Thiol/acrylate HA reaction). - Rapid development of amylase-expressing spheroids. 88
- Human SG stem/progenitor cells (hS/PCs). - Spheroids respond to TGF-βsignaling and improve K7 expression in hS/PCs.
- HepatoSTIM medium.
h
- 3D culture maintained for up to 14 days.
Bioactive basement membrane-
derived peptides conjugated HA
- Cells embedded into precursor before gelation (Thiol/acrylate HA reaction). - Peptide-modified HA promotes cell self-assembly into multicellular spheroids with close cell−cell
interaction.
89
- hS/PCs.
- HepatoSTIM medium.
i
- RGDSP and TWSKV-modified HA gels accelerate cell proliferation.
- 3D culture maintained for 14 days. - RGDSP-HA gels produce large spheroids.
- Cells embedded into precursor before gelation (Thiol/acrylate HA reaction). - HA-based 3D culture enhanced de expression of progenitor SG markers, proliferation, and viability
of hS/PCs.
63
- hS/PCs. - hS/PCs differentiate into acinar-like lineage under β-adrenergic and cholinergic agonist induced
stimulation.
- HepatoSTIM medium.
i
- 3D culture maintained for 20−25 days.
Decellularized extracellular matrix
(dECM)
- Rat submandibular gland chemical decellularization (rdECM-SMG) by detergent
immersion.
- Maintenance of ECM and basement membrane proteins in the rdECM-SMG, supports cell
adhesion.
90
- Cell reseeding under rotatory cell culture system. - Expression of key SG proteins after cell reseeding into rdECM-SMG.
- Rat primary SMG cells.
- Mammary epithelial growth medium.
- Bioengineered system maintained for 14 days.
- rdECM-SMG decellularization by detergent immersion. Cells embedded in gel. - rdECM-SMG-hydrogel-embedded rSGSCs survive and express SG functional differentiation
markers.
91
- Rat SG stem/progenitor cells (rSGSCs). - Increase in alpha-amylase levels and decrease in adult ductal stem/progenitor markers in 3D
cultures.
- DMEM: F12.
f
- Comparable levels of epithelial TJs markers to native SG.
- 3D culture organoids maintained for 5 days.
- Porcine SMG chemical decellularization (pDSG). Cell encapsulation in gel precursor. - pDSG-gel preserved ECM components and secreted factors. 92
- Primary SMG mesenchymal stem cells (SGMSCs). - pDSG-gel promote SGM- SCs migration and recruitment through the activation of PI3K/AKT
signaling pathway.
-α-MEM.
k
- Injected acellular pDSG-gel recruited endogenous cells and promote the formation of acinar and
ductal-like structures in the rat’s injury sites.
- Cultures maintained for up to 7 days. - pDSG-gel suppress fibrogenesis within the injured SG tissues.
Synthetic
Matrix Metalloproteinase-
degradable polyethylene glycol
(MMP-PEG)
- Mixing precursors and photo cross-linking. - Expression of SG markers in spheroids. 58
- Cell-laden gel formation using 1 mL syringe. - Maintenance of secretory protein expression.
- Mice cell clusters. - Acinar-to-ductal metaplasia observed in the spheroid produced in the hydrogel.
- DMEM: F12.
g
- Cultures maintained for up to 14 days.
MMP-PEG in
polydimethylsiloxane (PDMS)
microbubble (MB) array
- Mixing precursors with cells, loading them onto a PDMS/MB array, and photo cross-
linked.
- Viable mice and human salivary microtissue formation using MMP-PEG-encapsulated cell clusters
in MB array.
70
- Human and murine AIDUCs. - Expression of key salivary gland markers.
- DMEM: F12.
g
- Polarized localization of functional proteins.
- Cultures maintained for up to 14 days. - Calcium signaling agonists’ response and secretion of salivary proteins.
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cell−cell and cell−ECM interactions in 3D. This is especially
important since SG epithelial/acinar cells must receive
spatiotemporally controlled cues to allow apicobasal polar-
ization necessary for formation of functional 3D microtissues.
PEG-based polymers have shown the most promising results in
maintaining cell polarization and functional properties of ASC-
derived SG organoids.
106
However, there is still a gap in
optimizing synthetic matrices for engineering complex SG
models supported by non-SG cells that require additional
natural or synthetic ECM derivatives to function unanimously.
We previously compiled a description of the dierent types of
natural and synthetic hydrogels used for SG tissue engineering
applications.
34
Figure 2 illustrates examples of currently used
biomaterial strategies using natural and synthetic biomaterials
in engineering 3D SG models.
2.3. Bioprinting Strategies toward Salivary Gland 3D
Culture. 3D-bioprinting strategies have been rapidly growing
and are widely explored in the field of tissue engineering and
regenerative medicine.
107
While 3D bioprinting strategies are
still at its infancy in the context of bioengineering SGs, the first
evidence of bioprinted SG spheroids was described using a
magnetic 3D bioprinting (M3DB) strategy.
68
Using this
technique, human dental pulp stem cells (hDPSCs) were
first tagged with magnetic nanoparticles for 12 h, followed by
seeding into 96-well low attachment plates. Tagged cells in the
microplates were subjected to gravity and centrifugation
supported by a magnetic pin drive underneath the plates to
force cells into spheroids. These spheroids were then treated
with FGF10 to induce SG epithelial dierentiation followed by
characterization and transplantation into ex vivo models.
68
Recently, Yin et al. used a microfluidic, coaxial 3D printing
technique to fabricate thin cell-laden hydrogel fibers and
hollow microtubes to recapitulate SG epithelium and
branching characteristics in vitro.
108
The authors reported
that cultured SG cells maintain viability and retained
phenotype within both the bioprinted hydrogel fibers and
the tubes. Such an approach can be potentially used to perform
compartmentalized biofabrication of SG components, such as
an acinar and a ductal unit toward a fully functional gland
formation.
2.4. Tuning ECM Analogues for Salivary Gland 3D
Culture. The current SG bioengineering niche focuses on
utilizing hydrogels as essential ECM components for the
scaold-based creation of SG spheroids and organoids.
Complementary to matrix/scaold-based culture systems,
scaold-free approaches like suspension or microwell-based
cultures and bottom-up strategies contribute to scalable and
clinically applicable SG organ development for use in gland
regeneration. The shift away from animal-derived matrices has
prompted the exploration of alternative biomaterials such as
alginate, HA, and PEG in hydrogels for a 3D SG cell culture.
However, while hydrogel-based ECMs are attractive options to
culture SG cells in 3D, the biomechanical properties of these
ECM analogues need optimization. The matrix stiness,
architecture, and degradation properties of hydrogels should
be tuned to match the features of the native SG tissue
microenvironment.
34
These are crucial parameters that aect
the maintenance of the secretory cell phenotype and function
in long-term 3D cultures. Several studies have documented the
approximate stiness values of normal (∼9−33 kPa) and
radiation-injured (mean ∼23 kPa) human SG tissues using
shear wave elastography (SWE) techniques.
109,110
Similarly,
2D SWE of PA and SMGs of primary Sjogren’s patients
Table 1. continued
Biomaterial Cells, 3D system, and culture conditions Key Findings ref
PEG and polycaprolactone (PCL) - Photopatterning of PEG hydrogel in the presence of an electrospun PCL nanofibrous
scaffold.
- PCL microwells promote cell aggregation to form 3D acinar-like spheroids. 50
- human parotid epithelial cells (hPECs). - High expression of epithelial markers, TJs, AJs, and cytoskeleton proteins.
- Keratinocyte-serum-free.
j
- High levels of alpha-amylase and intracellular calcium concentration.
- Cultures maintained for up to 5 days.
- Photolithography of PEG hydrogel in the presence of an electrospun PCL nanofibrous
scaffold. Cells seeded on top of precoated plates.
- SGSC spheroids express higher stem cell/pluripotent markers than 2D monolayer cultures. 93
- Human SGSC. - Enhanced differentiation into SG cells and paracrine secretion compare with 2D culture.
- DMEM.
l
- Enhanced radioprotective properties in SGSCs spheroids.
- Cultures maintained for up to 7 days.
a
Supplemented with: Epithelial growth factor (EGF), fibroblast growth factor-2 (FGF-2), N2 supplement, insulin, dexamethasone, fetal bovine serum (FBS), ROCK inhibitor Y-27632.
b
Horse serum,
penicillin, streptomycin, cholera toxin, hydrocortisone, human insulin, bFGF/FGF2.
c
Fetal calf serum, penicillin, streptomycin, Glutamax, EGF, FGF-2, N2 supplement, insulin, dexamethasone.
d
FBS,
penicillin, streptomycin, FGF2, or EGF.
e
FBS, retinoic acid, triiodothyronine, EGF, glutamine, insulin, transferrin, sodium selenite, hydrocortisone, gentamicin.
f
Embryonic stem cell qualified FBS,
penicillin, streptomycin, nicotinamide, insulin, transferrin, selenium supplement, dexamethasone, β-mercaptoethanol, mouse EGF receptor.
g
Glutamine, antibiotic/antimycotic, N2 supplement, insulin,
dexamethasone, EGF and/or bFGF.
h
Penicillin, streptomycin, amphotericin B, EGF.
i
Penicillin, streptomycin, fungizone, EGF.
j
EGF, calcium chloride, antibiotics.
k
FBS, penicillin, streptomycin.
l
Low
glucose, FBS, penicillin, streptomycin.
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Figure 2. Examples of biomaterial strategies used in developing SG 3D models. A) Preparation of fibrin hydrogels using L1-peptide conjugated
fibrinogen. Reproduced from 85. Copyright 2016 American Chemical Society. B) Chemical configurations of HA-SH, HA-AES (mono-2-
(acryloyloxy) ethyl succinate), and MI (maleimide) functionalized peptides (top). Final matrix was prepared by first conjugating the peptides to
HA by thiol−maleimide reaction, followed by addition of HA-AES to from covalent networks through thiol−acrylate reactions (bottom).
Reproduced from 89. Copyright 2021 American Chemical Society. C) Schematic illustration of steps involved in the fabrication of PCL
nanofibrous scaolds with PEG wall micropatterning. Briefly, 800-nm-thick PCL fibers with 50 μm thickness were fabricated using electrospinning.
The nanofiber matrix was oxygen plasma-treated, and microwell-shaped hydrogel micropatterns, using a hydrogel precursor solution, were created
on the PCL nanofibers using photolithography and UV light. These PEG hydrogel-covered microwells supported by PCL nanofibers were further
used for cell seeding and spheroid formation. Reproduced with permission from 50. Copyright 2016 Elsevier. D) Lyophilized nonbornene-
functionalized 4-arm PEG macromer, an MMP degradable peptide sequence, 0.05 wt % LAP photoinitiator, and 100 μg/mL laminin are mixed to
form the hydrogel precursor. This precursor is then mixed with SG cell clusters and photopolymerized at 5 mW/cm2, 365 nm using UV light for 5
min to encapsulate cells into hydrogels for downstream culture and analysis. Reproduced with permission from 106. Copyright 2022 Wiley. E)
Photographs representing sequential steps in the establishment of porcine decellularized SG matrix as scaolds for SG 3D culture. Reproduced with
permission from 92. Copyright 2023 Elsevier.
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I

showed significantly higher values than controls depending on
the disease severity.
111
Clinical data on injured SGs of patients suering from
xerostomia due to dierent conditions can provide a
framework for the fabrication of ECM mimetics tuned to
recapitulate in-vivo-like conditions. Nevertheless, it is still
essential to develop a system that is adaptable to in vitro/ex
vivo cultures, as they may vary significantly from the native
environment depending on the scaolds and cell source used.
For example, Peters et al. developed polyacrylamide gels to
evaluate the growth and development of mouse embryonic
SMGs and found that softer substrates (0.48 kPa) promoted
expansion, expression of key SG markers, and branching
morphogenesis in developing SMG which was altered when
cultured in stier substrates (19.66 kPa).
112
More recently, slow degradability of ECM hydrogels has
been attributed to promoting maintenance of adult SG
phenotype and function in microculture systems.
113
Addition-
ally, SGs develop through branching morphogenesis, a key
process which requires specific ECM components (for
example: laminin, fibronectin, etc.) within permissible matrix
to allow coordinated signaling for cell proliferation, migration,
and reorganization.
114
Thus, several studies focus on the use of
cell binding peptides or motifs to natural ECM components to
not only tune hydrogel stiness, but allow SG cells to
reorganize into like native tissues.
86,89
Studies conducted on
developing SGs also provide evidence on the importance of
strong cell−ECM and weak cell−cell interactions as pivotal
cues for branching morphogenesis and cellular reorganiza-
tion.
115
In summary, future biomaterial advances for SG tissue
engineering need focused exploration of ECM compositions
necessary to support in vitro secretory function of SG cells in
the long term. In addition, establishment of versatile, tunable,
and clinically translatable hydrogels will pave the way for
development of salivary organoids for stem cell therapy and
organ transplantation. Further, the integration of additive
manufacturing techniques, along with hydrogel-based ECM
derivatives, can provide a complex microenvironment con-
ducive to the long-term establishment of ex vivo SG models
featuring vascular and neural components toward the develop-
ment of artificial salivary glands.
3. TRANSLATING THE BIOENGINEERED SALIVARY
GLANDS�CURRENT APPLICATIONS
The surge in 3D organoid technology research is notable,
driven by its capacity to replicate the cellular, extracellular, and
functional attributes of organs or tissues. While organoid
systems like intestinal, brain, kidney, etc., to name a few, have
reached established stages, SG organoid culture, particularly
PSC-based organoids, is still in the early phases of exploration.
Nevertheless, there have been promising outcomes in the
application of SG organoid technology for various purposes.
This section will delve into the two primary applications of SG
organoids: in vitro disease modeling and in vivo trans-
plantation.
3.1. Bioengineering 3D Salivary Gland Models for
Disease Modeling. For numerous decades, in vitro research
has predominantly relied on the utilization of simple cell lines
and 2D cultures. While this foundational research has been
successful in unravelling disease mechanisms and identifying
therapeutics, contemporary investigations aim to achieve
similar outcomes using more intricate physiological models,
such as 3D cultures, thereby expediting clinical translation. SG
spheroids and organoids have become regular tools in the
scientific arsenal and routinely employed to explore SG
development, physiology, and responses to injury.
Nagle et al. conducted an assessment of the in vitro radiation
response of the HSG cell line in both 2D and 3D cultures,
employing varying linear energy transfer (LETs).
116
In the 2D
culture, a dose-dependent response to LET irradiation was
observed, whereas the 3D culture exhibited a reduced
sensitivity to irradiation. Additionally, higher LETs were
associated with increased expression of the DNA damage
response marker γH2AX under both 2D and 3D conditions.
Furthermore, the study explored the impact of SG stem cells in
3D cultures under escalating LETs, revealing that stem cells
demonstrated greater radioresistance compared to the HSG
cell line.
116
However, in a later study by the group, they
determined that at low doses of irradiation, the SG stem cells
become hypersensitive due to a lack of DNA Damage
Response (DDR) activation which is activated at higher
doses that illicit appropriate DDR.
117
This response was tested
in both murine and human SG and thyroid gland organoid
models and thus serves as clinically relevant factors when
considering organ preservation strategies during radiation
therapy.
Elsewhere, Shin et al. established an organotypic spheroid
model to simulate radiation injury in SGs.
118
They isolated
epithelial cells from the human parotid (PA) gland and
cultured them on plastic or Matrigel, examining the impact of
10 and 20 Gy irradiation on cell phenotype. The irradiated SG
spheroids exhibited a dose-dependent reduction in gland-
specific markers and functionality. Notably, the SG spheroid
model demonstrated enhanced tracking of changes in Ca2+
activity following irradiation compared to 2D monolayer
culture.
118
The same research group developed a spheroid
coculture model comprising human PA gland spheroids and
human adipose-derived mesenchymal stem cells (huMSCs)
preconditioned under normoxia (huMSCNMX) or hypoxia
(huMSCHPX).
119
In vitro findings revealed that the presence
of huMSCHPX significantly enhanced radioresistance in PA
gland-derived spheroids while preserving acinar cell integrity.
FGF10, secreted as a paracrine factor, emerged as a crucial
contributor to radioprotection. This observation was further
validated in vivo, where the transplantation of huMSCHPX
into irradiated mice successfully rescued salivary gland (SG)
function.
119
An indirect method to investigate factors rescuing
SG function was to evaluate the extent of salisphere formation
from irradiated glands. Nguyen et al. showed that irradiated
mice SGs led to fewer salisphere formation in vitro which
improved proportionally with increasing FBS concentration.
Further, the group found that irradiated mice when injected
with insulin like growth factor (IGF-1), improved SG function
and yielded better salisphere formation in vitro.
120
Similarly,
Yoon et al. investigated the impact of hepatocyte growth factor
(HGF) in restoring impaired SG function postirradiation,
highlighting the presence of HGF receptor, MET in adult
SGs.
121
Treatment of human PA gland organoids with HGF
following radiation exposure resulted in enhanced SG markers
and improved secretory functions. Moreover, the study found
that the radioprotection achieved through HGF treatment was
reversed when PA gland organoids were treated with a MET
inhibitor.
121
These studies show the significance of in vitro
models that allows exploration of therapeutic targets for
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treating SG hypofunction further in in vivo preclinical and
human clinical studies.
Recent progress in microfabrication techniques has facili-
tated their integration into hydrogels for tissue engineering.
Song et al. engineered a platform consisting of a 200 μm
diameter cylindrical microbubble (MB) array that was
integrated into a 48-well plate system.
70
Isolated acinar−
ductal cell clusters were combined with MMP-degradable PEG
hydrogels and cultured in these MB arrays to evaluate the
maintenance of the SG phenotype and secretory function. The
researchers further refined this tissue chip as a drug screening
platform and investigated the impact of radioprotective agent
Figure 3. Transplantation strategies using bioengineered SGs. A) Schematic illustration showing steps for the orthotopic transplantation of iSGs in
PA gland defective mice. B) From left−right, photograph of GFP positive iSGs visualized in mice after transplantation, scale: 500 μm; H&E
staining of the iSG, scale: 500 μm; PAS staining of the iSG, scale: 50 μm; fluorescence images showing GFP positive structures at the transplanted
site and integrated with host gland structures; scale: 200 μm. C) Immunofluorescence staining showing key SG specific markers in iSG transplanted
without mesenchyme; scale: 50 μm. Reproduced from reference 124. Available under a CC-BY 4.0 (http://creativecommons.org/licenses/by/4.0/
) license. Copyright 2018 Tanaka et al. D) Schematic illustration of human SMG epithelial stem/pluripotent cells (hSMGepiS/PCs) reconstitution
and heterotopic transplantation. E) Top: Photographs of mice kidneys transplanted with hSMGepiS/PC derived spheres, in combination with
E12.5SMG mesenchyme or mesenchyme alone, bottom: H & E staining of showing the transplanted sections. Scale: 4 mm. F) and G)
Immunofluorescence staining of transplanted spheres alone or in combination with mesenchyme shows acinar (AQP5), epithelial (E-cadherin) and
ductal (K19) markers. Scale: 50 μm. Reproduced from reference 62. Available under a CC-BY 4.0 (http://creativecommons.org/licenses/by/4.0/)
license. Copyright 2020 Sui et al.
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WR-1065 on irradiated SG mimetics. DDR was evaluated on
the chip using live/dead assays and immunostaining of DNA
damage markers γH2AX and 53BP1.
70
The study results
support the utility of tissue chip arrays in combination with
hydrogels for maintaining the SG culture and their potential
application in drug screening.
SG organoids have also emerged as promising models for
the study of viral infection and replication that aect SGs. In a
comprehensive study conducted by Tanaka et al., the
researchers successfully established and characterized human
induced pluripotent stem cell-derived SG (hiSG) organoids.
75
These hiSG organoids were utilized to investigate SG
development as well as the infection and replication dynamics
of SARS-CoV-2. The hiSGs exhibited properties resembling
those of embryonic SGs, comprising a heterogeneous group of
cells that supported normal SG development. Upon inocu-
lation with the SARS-CoV-2 virus, the hiSG organoids
demonstrated virus uptake and subsequent replication,
indicating their potential as novel models for studying viral
infections, particularly those harboring in SGs and spreading
via saliva.
75
In a similar approach using murine salivary gland
(SG) salispheres and human SG cell lines, Ghosh et al.
explored the potential of enteric viruses to infect SGs.
122
Mice
SG salispheres, when inoculated with rotavirus and MNV-1,
exhibited a 10-fold increase in viral genomic RNA and the
presence of MNV-1 in the culture supernatant, respectively.
Additionally, human SG cell lines cultured in a 2D monolayer
were utilized to assess the ecacy of noroviruses in infecting
and replicating within SG cells. The results indicated that,
following inoculation and sequential passaging at P4, there was
a 100-fold increase in intracellular viral titers and a 10-fold
increase in extracellular viral titers.
122
SG 3D models have proven instrumental in disease
modeling, oering a physiologically relevant platform to
study various conditions aecting these glands. They provide
a 3D microenvironment that better recapitulates the complex-
ity of the native tissue, allowing for an accurate representation
of disease processes. For instance, prolonged culture of SG
organoids and spheroids allows examination of both immediate
and delayed eects of radiation injury at dierent time points,
providing insights at both the cellular and molecular levels.
Such investigations may uncover novel biological pathways
that could be targeted to mitigate radiation-induced damage to
diverse cell populations within SGs.
Functional 3D SG models, when integrated with high-
throughput platforms, can be employed to screen large libraries
of drugs or small molecules for the treatment of xerostomia.
These investigations, if conducted thoroughly, can significantly
expedite the drug discovery pipeline and reduce the associated
costs. SG microtissues are versatile tools that can also be
combined with nonparenchymal cell types (vascular, stromal,
neural) to explore dynamic cellular interactions and multiomic
changes in response to radiation therapy, disease progression,
and treatments. Furthermore, 3D cell models oer oppor-
tunities to introduce various genetic perturbations for studying
genes contributing to autoimmune conditions like Sjogren’s
Syndrome. In summary, SG spheroids and organoids can oer
a bridge between traditional 2D cultures and in vivo studies,
allowing researchers to gain deeper insights into the
mechanisms of diseases and test potential therapeutic
interventions.
3.2. In Vivo Transplantation of Bioengineered
Salivary Glands. In recent years, the exploration of organ
transplantation for irreversibly injured SGs has gained much
attention. Several studies have explored strategies to transplant
specific SG stem cells or epithelial cells into damaged glands to
revive lost SG function. However, while replenishing injured
stem/progenitor cells or epithelial secretory cells may oer
partial improvement in salivary flow,
36,60
it may not
compensate for chronic injury or severe secretory loss. Such
clinical conditions necessitate the need for SG replacement
through bioengineered glands to sustain saliva secretion.
ASC-derived salispheres and salispheres-derived stem cells
have shown the ability to rescue SG hypofunction caused due
to radiotherapy.
61,123
Shin et al. conducted a comparative
analysis of the eectiveness of SG stem cells cultured in either
2D or Matrigel in enhancing gland function postradiation.
69
WNT-primed SG stem cells cultured in 2D or 3D were
transplanted into mice 4 weeks after radiation exposure. The
engraftment of stem cells was assessed at weeks 2 and 12 post-
transplantation. The findings indicated that 3D-cultured stem
cells exhibited superior grafting within the mouse SG glands
compared to their 2D counterparts. Moreover, histological
assessment of glands transplanted with 3D-cultured cells
revealed a higher population of mucin-producing secretory
cells and lower levels of fibrosis compared to those
transplanted with 2D-cultured cells. Mice transplanted with
3D stem cells also demonstrated an improved salivary flow
rate.
69
These outcomes oered initial evidence suggesting that
3D-cultured SG stem cells may preserve or enhance their
regenerative potential when transplanted, yielding superior
results compared with single-cell transplantation cultured in
2D conditions.
Tanaka et al. pioneered the initial exploration of orthotopic
transplantation of ex-vivo-cultured SGs.
124
SGs organoids
derived from mouse ESCs, either alone or previously
cocultured with mesenchyme derived from embryonic SMGs,
were transplanted into a mouse model deficient in PA glands.
The induced SGs (iSGs) were positioned in a collagen gel
drop with a poly(glycolic acid) (PGA) monofilament serving
as a guide. This PGA monofilament was inserted into the
masseter muscle, acting as a guide to integrate the ex-vivo-
cultured iSGs into the PA duct region (Figure 3A). The entire
construct was then secured using an 8−0 nylon thread.
124
The
transplanted iSGs exhibited physiological development in vivo,
forming connections with the host PA duct and displaying
positive populations of acinar and ductal cells (Figure 3B).
Immunofluorescence staining revealed the expression of gland-
specific markers (AQP5, K18, K5, SMA, and NKCC1) and
secretory markers (mucin and amylase), resembling normal
glands (Figure 3C). Moreover, the transplanted iSGs, whether
alone or in combination with embryonic SMG-derived
mesenchyme, established connections with nerve fibers,
expressing the nerve fiber marker TUBB3 and the endothelial
marker CD31. Collectively, the authors concluded that ESC-
derived iSGs can integrate into normal recipient SGs upon
transplantation and undergo maturation akin to embryonic SG
development in vitro.
124
A recent study by Zhang et al. presented an innovative
approach for generating SG placodes from PSCs and explored
their potential for in vivo transplantation.
79
In a sequential
process, the authors first dierentiated mouse embryonic stem
cells into an oral ectoderm using bone morphogenetic protein
4 (BMP4). Subsequently, retinoic acid and bFGF were added
to create SG placodes, which were transplanted into the renal
capsules of the mice. Results from the study indicate that
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transplanted placodes at day 30 developed and dierentiated
into ductal components of the SGs capable of forming
lumens.
79
Specifically, they exhibited populations of striated
and excretory ductal cells; however, the SG placodes lacked
acinar-specific cell populations. Although PSC-derived SG
organoids are still in the early stages of development, they have
demonstrated significant potential for dierentiation and
regeneration in dierent compartments of native SG tissue.
A heterotopic transplantation approach utilizing ASC-
derived SG organoids, in combination with mouse embryonic
SG mesenchyme, has been proposed as a promising method
for SG organ regeneration.
62
These SG organoids induced by
FGF10 exhibited markers indicative of the SG phenotype,
along with functional characteristics. SG spheres, either alone
or reconstituted with mesenchyme, were isolated from mice
D12.5 embryonic SMG and were transplanted into the renal
capsule of mice (Figure 3D, E). At day 30, the analysis of
spheres transplanted alone revealed the maintenance of gland-
specific markers such as SMA and K19, but poor aquaporin 5
(AQP5) expression, possibly attributed to the lack of FGF10-
induced stimulation in vivo
62
(Figure 3E, F). More recently,
SG-specific ECM was used to transdierentiate rat BM-derived
MSC (BM-MSCs) into SG-like organoids when transplanted
into renal capsule of mice.
125
The authors showed that
transdierentiated cells show SG-specific markers when
cultured in vitro and organoid-like characteristics when
transplanted into mice. The study reported that BMMSCs
could be utilized as an alternative and ample source to generate
bioengineered SGs for use in transplantation.
125
Leveraging the
self-renewal capacity and lineage specificity of SG tissue-
resident stem/progenitor cells, Xu et al. employed organoids
derived from Sox9+ cells to rescue radiation-injured SGs.
126
Around 200 organoids sourced from Tomato+ mice SMGs
were injected into the irradiated SMGs of wild-type mice. The
Tomato+ organoids were observed to be distributed in both
the acinar and ductal compartments of the SMGs.
Furthermore, the salivary flow rate in mice that received
organoid transplants was higher than nontreated mice,
suggesting the potential of bioengineered SG organoids in
replacing or regenerating functionally relevant SG struc-
tures.
126
Collectively, these studies provide reassurance
regarding the potential of PSC- and ASC-derived SG
microtissues, with supporting mesenchyme, to facilitate SG
regeneration after injury. Still, thus far, only a handful of
studies have successfully shown SG organoid or spheroid
transplantation in the in vivo models.
5. CONCLUSIONS
The recent success in bioengineering functional SGs models
can be directly attributed to ongoing advancements in SG 3D
culture techniques, enabling the generation of physiologically
relevant SG microtissues. These 3D models, whether in the
form of microtissues, spheroids, or organoids, have demon-
strated immense potential to maintain functional phenotypes,
cell heterogeneity, and genomic stability in vitro. Importantly,
these in vitro successes attest that they hold great promise for
in vivo transplantation, providing a potential solution for the
replacement of damaged or nonfunctional salivary glands. The
progress in SG 3D cultures is a key enabler in the development
of bioengineered SGs, bringing us closer to achieving
functional and clinically applicable solutions for SG replace-
ment. Nonetheless, several challenges still exist, especially in
terms of how these 3D cell models are produced, the problems
associated with their size and long-term maintenance, and the
suitability of their usage for clinical applications.
While certain growth factors and cytokines have been
identified as essential soluble cues in SG bioengineering, no
one culture medium, in terms of composition or concentration,
can be applied across SG 3D cell culture systems. This is
especially important when considering the mechanics of the
matrix, their variable composition, and the resultant size of the
3D cell model. Use of animal-derived ECM components like
Matrigel poses significant regulatory hurdles for future clinical
translation. Additionally, while animal-derived cells serve as
easy sources to optimize new 3D culture systems, extensive
validation of these systems using human SG cells is imperative
for their prospective use in aected patients. Alternative
sources like natural hydrogels, dECMs, or their combinations
are fast emerging, hold great promise and can be combined
with human SG cells to validate novel in vitro and ex vivo
models.
Despite these advances, the size of the SG spheroids and
organoids obtained from these 3D culture systems often
becomes the limiting factor when contemplating their clinical
suitability for organ replacement therapies. Although organ
germ methods have been proposed to alleviate this challenge,
the practicality of the approach in humans has yet to be
evaluated. Even with functional and sizable organoids,
concerns revolve around the presence of the right type of
stem cell in an organoid and their stemness especially after
transplantation, which is crucial during organ replacement.
Additionally, integration of these ex vivo developed 3D
microtissues into existing normal gland needs exploration
and targeted approaches to achieve function, especially with
respect to presence of neural and vascular components. In the
future, progress in rapid prototyping, bioprinting, and
microfluidic technologies is anticipated to enable the
integration of various cell types, encompassing salivary,
vascular, neural, and immune cells, in the pursuit of SG
organ engineering.
■AUTHOR INFORMATION
Corresponding Author
Simon D. Tran −McGill Craniofacial Tissue Engineering and
Stem Cells Laboratory, Faculty of Dental Medicine and Oral
Health Sciences, McGill University, Montreal, QC H3A 0C7,
Canada; orcid.org/0000-0001-5594-359X;
Email: simon.tran@mcgill.ca
Authors
Sangeeth Pillai −McGill Craniofacial Tissue Engineering and
Stem Cells Laboratory, Faculty of Dental Medicine and Oral
Health Sciences, McGill University, Montreal, QC H3A 0C7,
Canada; orcid.org/0000-0002-4475-984X
Jose G. Munguia-Lopez −Department of Mining and
Materials Engineering, McGill University, Montreal, QC
H3A 0C5, Canada; orcid.org/0000-0002-5173-6494
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsabm.4c00028
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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https://doi.org/10.1021/acsabm.4c00028
ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
M

Funding
This manuscript is supported by the Natural Sciences and
Engineering Research Council of Canada (NSERC) grant
#03615. The first author is supported in part by the funding
from the Fonds de recherche du Quebec−Sante(FRQS)
doctoral award #304367.
Notes
The authors declare no competing financial interest.
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