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Citation: Willemse, J.; van der Laan,
L.J.W.; de Jonge, J.; Verstegen, M.M.A.
Design by Nature: Emerging
Applications of Native Liver
Extracellular Matrix for
Cholangiocyte Organoid-Based
Regenerative Medicine.
Bioengineering 2022,9, 110.
https://doi.org/10.3390/
bioengineering9030110
Academic Editor: Joaquim M.
S. Cabral
Received: 17 January 2022
Accepted: 4 March 2022
Published: 7 March 2022
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bioengineering
Review
Design by Nature: Emerging Applications of Native Liver
Extracellular Matrix for Cholangiocyte Organoid-Based
Regenerative Medicine
Jorke Willemse , Luc J. W. van der Laan , Jeroen de Jonge †and Monique M. A. Verstegen *,†
Department of Surgery, Erasmus MC Transplant Institute, University Medical Center,
3015 CN Rotterdam, The Netherlands; j.willemse@erasmusmc.nl (J.W.); l.vanderlaan@erasmusmc.nl (L.J.W.v.d.L.);
j.dejonge.1@erasmusmc.nl (J.d.J.)
*Correspondence: m.verstegen@erasmusmc.nl; Tel.: +31-10-703-5528
† These authors contributed equally to this work.
Abstract:
Organoid technology holds great promise for regenerative medicine. Recent studies show
feasibility for bile duct tissue repair in humans by successfully transplanting cholangiocyte organoids
in liver grafts during perfusion. Large-scale expansion of cholangiocytes is essential for extending
these regenerative medicine applications. Human cholangiocyte organoids have a high and stable
proliferation capacity, making them an attractive source of cholangiocytes. Commercially available
basement membrane extract (BME) is used to expand the organoids. BME allows the cells to self-
organize into 3D structures and stimulates cell proliferation. However, the use of BME is limiting
the clinical applications of the organoids. There is a need for alternative tissue-specific and clinically
relevant culture substrates capable of supporting organoid proliferation. Hydrogels prepared from
decellularized and solubilized native livers are an attractive alternative for BME. These hydrogels
can be used for the culture and expansion of cholangiocyte organoids in a clinically relevant manner.
Moreover, the liver-derived hydrogels retain tissue-specific aspects of the extracellular microenviron-
ment. They are composed of a complex mixture of bioactive and biodegradable extracellular matrix
(ECM) components and can support the growth of various hepatobiliary cells. In this review, we
provide an overview of the clinical potential of native liver ECM-based hydrogels for applications
with human cholangiocyte organoids. We discuss the current limitations of BME for the clinical
applications of organoids and how native ECM hydrogels can potentially overcome these problems
in an effort to unlock the full regenerative clinical potential of the organoids.
Keywords:
extracellular matrix; cholangiocyte organoids; bile duct; liver; tissue engineering;
regenerative medicine; culture substrates
1. Introduction
The shortage of donor organs is a central theme in the field of liver transplantation,
which still is the only curative treatment option for patients suffering from end-stage liver
failure. The donor shortage leads to high waiting list mortality. However, suitable donor
livers often do not become available in time for up to 20% of the patients on the waiting
list [
1
–
3
]. Efforts made to increase the pool of available donor livers include the use of
extended criteria donor organs, such as the use of donation after circulatory death (DCD)
organs [
4
]. The use of DCD livers is associated with a higher incidence of ischemia-type
biliary lesions (16% vs. 3% when compared to donation after brain death) [
5
,
6
]. Cholangio-
cytes form an active barrier between the cytotoxic bile and surrounding tissue [
7
]. They
are sensitive to ischemia, and the extra period of warm ischemia in DCD transplantation
can cause deficits in the biliary epithelium, such as non-anastomotic strictures [
4
,
8
,
9
]. Ulti-
mately, 65% of patients with ischemic cholangiopathy require retransplantation, as there is
Bioengineering 2022,9, 110. https://doi.org/10.3390/bioengineering9030110 https://www.mdpi.com/journal/bioengineering
Bioengineering 2022,9, 110 2 of 17
currently no alternative treatment option available [
6
]. This does not only have far-reaching
impact on patients, but also reduces the number of available donor grafts for other patients.
Regenerative medicine strategies could repair the biliary deficits while the liver is
preserved ex vivo on an organ perfusion setup. Eshmuminov et al. recently showed
that it is feasible to maintain human livers on the pump for up to 7 days [
10
]. These
improvements in the field of organ preservation could open up a window of opportunity
for ex vivo organ repair. Stem cells or cholangiocytes can be used to repair deficits in the
biliary epithelium [11,12].
The aim of this review is to discuss the potential of native human liver extracellular
matrix (ECM) for the clinical grade applications of cholangiocyte organoids for regener-
ative medicine in patients. We discuss why the use of mouse tumor-derived basement
membrane extract (BME) is currently limiting the clinical applications of cholangiocyte
organoids. In addition, we provide an overview of requirements for clinically relevant
culture substrates. We will discuss how liver ECM hydrogels provide an alternative culture
substrate and why the use of decellularized liver tissue can unlock the full clinical potential
of cholangiocyte organoids.
2. The Potential of Organoids in Tissue Regeneration
The
in vitro
isolation and large-scale expansion of primary cholangiocytes cultured on
traditional cell culture plastic is challenging [
13
]. Alternative sources of cholangiocytes are
therefore required. Pluripotent stem cells (embryonic or induced) can be directed towards
cholangiocytes, but this requires extensive differentiation of cells [
14
–
16
]. Moreover, there
is the potential risk of aberrant (de)differentiation and teratoma formation [
17
–
19
]. The use
of adult tissue-specific progenitors to grow cholangiocyte organoids, enables cell expansion
from relatively small (0.5–1.0 cm
3
) human liver biopsies. These organoids maintain a
cholangiocyte-like phenotype in vitro [20].
The first cholangiocyte organoids derived from human liver biopsies were described
by Huch et al. in 2015 [
21
]. This research was based upon the discovery of Leucine-rich
repeat-containing G-protein coupled receptor 5 (LGR5) positive stem cells found in the
intestine [
22
], which gave rise to intestinal organoids [
23
]. A similar approach was described
for LGR5+ mouse liver-derived organoids [
24
], followed by human liver organoid cultures.
Huch et al. showed that EPCAM positive cells in human liver biopsies can give rise
to spheroid-like cultures with an efficiency of 28% (SD:
±
3%) [
21
]. These liver-derived
cholangiocyte organoids have a cholangiocyte-like phenotype and express cholangiocyte
markers, such as KRT-7/19 and EPCAM, as well as progenitor markers (e.g., SOX-9,
LGR-5
).
Moreover, they have a high and stable proliferation capacity. Relatively small tissue
biopsies can yield clinically relevant numbers of cells in a relatively short time span [
25
,
26
].
Moreover, the liver-derived organoids can express and upregulate hepatocyte markers,
such as albumin, HNF-4
α
and CYP-3A4, upon differentiation towards hepatocyte-like
cells [
21
,
26
,
27
]. This makes them a potential source of both cholangiocytes and hepatocytes.
However, the expression of hepatocyte markers and hepatocyte functionality does not yet
reach similar levels as primary human hepatocytes.
With the expansion of hepato-pancreato and biliary organoid research, a consistent
nomenclature was proposed, and the liver-derived organoids were renamed to intrahepatic
cholangiocyte organoids (ICO) to better reflect their origin [
20
] (see Figure 1for an overview
of the different cholangiocyte organoids and sources). Cholangiocyte organoids were estab-
lished from gallbladder tissue (gallbladder cholangiocyte organoids; GCO) [
28
], and from
the cholangiocytes’ inner lining the extrahepatic bile duct (EBD, extrahepatic cholangiocyte
organoids; ECO) [
27
,
29
]. Organoids were also initiated from the circulating EPCAM pos-
itive cells in fresh bile samples (bile-derived cholangiocyte organoids, BCO) [
30
]. These
organoids all share similar phenotypic features when cultured, as they all grow in similar
spherical structures with comparable proliferation rates [
27
,
30
,
31
]. They express similar
progenitor and mature cholangiocyte markers, but are uniquely related to their original
tissue of origin and show subtle differences. Rimland et al. showed that differences related
Bioengineering 2022,9, 110 3 of 17
to the regional origin of the organoids were retained between ICO, ECO and GCO [
31
]. In
addition, only ICO have the potential to upregulate hepatocyte markers when grown in
hepatocyte differentiation conditions [27].
Bioengineering 2022, 9, x FOR PEER REVIEW 3 of 17
They express similar progenitor and mature cholangiocyte markers, but are uniquely re-
lated to their original tissue of origin and show subtle differences. Rimland et al. showed
that differences related to the regional origin of the organoids were retained between ICO,
ECO and GCO [31]. In addition, only ICO have the potential to upregulate hepatocyte
markers when grown in hepatocyte differentiation conditions [27].
Figure 1. Schematic overview of the workup of cholangiocyte organoids in BME cultures and the
different sources of organoids. Cholangiocyte organoids can be initiated from liver tissue biopsies
(intrahepatic cholangiocyte organoids; ICO), extrahepatic bile duct tissue biopsies (extrahepatic
cholangiocyte organoids; ECO) and gallbladder tissue biopsies (gallbladder cholangiocyte organ-
oids; GCO). Organoids can also be initiated from bile samples (bile-derived cholangiocyte organ-
oids; BCO). Organoids are traditionally grown and expanded in BME.
3. Repairing Damaged Organs Using Cholangiocyte Organoids
The ability to generate large numbers of cells from relatively small (patient-derived)
biopsies is of interest for regenerative cell therapy applications. Therefore, cholangiocyte
organoid-derived cells could be a promising source of hepatobiliary cells for organ repair
applications [32–34]. ICO differentiated towards hepatocyte-like cells were capable of en-
grafting a damaged mouse liver. These cells showed some level of hepatocyte functional-
ity as human albumin was detected 120 days after engrafting [21]. However, engraftment
rates were extremely low (<1%). Therefore, it is not likely that ICO will be used as a cell
source for hepatocyte transplantation, as long as current hepatocyte differentiation proto-
cols and cell administration protocols are not improved.
Cholangiocyte organoids can also yield cholangiocyte-like cells without the need for
additional differentiation steps. Therefore, these cells are of interest for the repair of dam-
aged biliary epithelium. Recently, ground-breaking proof of concept was given by Sam-
paziotis et al. They showed that cholangiocyte organoids can successfully repair deficits
in the biliary epithelium of intrahepatic bile ducts after injecting cells that were derived
from the organoids into the biliary tree of a mouse model (Figure 2) [35]. They also showed
that GCO can be used to repair deficits of the biliary epithelium of human livers ex vivo
while the liver was perfused on a normothermic machine perfusion device. This shows
the enormous clinical potential of cholangiocyte organoids.
Figure 1.
Schematic overview of the workup of cholangiocyte organoids in BME cultures and the
different sources of organoids. Cholangiocyte organoids can be initiated from liver tissue biopsies
(intrahepatic cholangiocyte organoids; ICO), extrahepatic bile duct tissue biopsies (extrahepatic
cholangiocyte organoids; ECO) and gallbladder tissue biopsies (gallbladder cholangiocyte organoids;
GCO). Organoids can also be initiated from bile samples (bile-derived cholangiocyte organoids; BCO).
Organoids are traditionally grown and expanded in BME.
3. Repairing Damaged Organs Using Cholangiocyte Organoids
The ability to generate large numbers of cells from relatively small (patient-derived)
biopsies is of interest for regenerative cell therapy applications. Therefore, cholangiocyte
organoid-derived cells could be a promising source of hepatobiliary cells for organ repair
applications [
32
–
34
]. ICO differentiated towards hepatocyte-like cells were capable of en-
grafting a damaged mouse liver. These cells showed some level of hepatocyte functionality
as human albumin was detected 120 days after engrafting [
21
]. However, engraftment rates
were extremely low (<1%). Therefore, it is not likely that ICO will be used as a cell source
for hepatocyte transplantation, as long as current hepatocyte differentiation protocols and
cell administration protocols are not improved.
Cholangiocyte organoids can also yield cholangiocyte-like cells without the need
for additional differentiation steps. Therefore, these cells are of interest for the repair
of damaged biliary epithelium. Recently, ground-breaking proof of concept was given
by Sampaziotis et al. They showed that cholangiocyte organoids can successfully repair
deficits in the biliary epithelium of intrahepatic bile ducts after injecting cells that were
derived from the organoids into the biliary tree of a mouse model (Figure 2) [
35
]. They also
showed that GCO can be used to repair deficits of the biliary epithelium of human livers
ex vivo while the liver was perfused on a normothermic machine perfusion device. This
shows the enormous clinical potential of cholangiocyte organoids.
Bioengineering 2022,9, 110 4 of 17
Bioengineering 2022, 9, x FOR PEER REVIEW 4 of 17
Figure 2. Cholangiocyte organoids can repair deficits in the biliary epithelium. This treatment can
restore adequate drainage of bile and prevent build-up of toxic bile inside the liver. Cholangiocyte
organoids can be infused into the biliary tree to repair the damaged biliary tree.
4. Basement Membrane Extract as Culture Substrates
The clinical application of cholangiocyte organoids is currently limited by the use of
non-GMP (good manufacturing practice)-compliant basement membrane extracts (BMEs)
in which organoids are typically cultured. BME is a complex mixture of extracellular ma-
trix (ECM) components derived from the tumor mass produced by Englebreth-Holm-
Swarm (EHS) mouse cells. These cells produce an abundance of basement membrane
components, which can be extracted and processed into hydrogels [36,37]. The main con-
stituents of BME are laminin-111, collagen type IV and enactin [37–39]. Other components
include a myriad of other bioactive ECM components and growth factors [39]. The extrac-
tion of these components and subsequent reconstitution was first described in 1986 by
Kleinman et al. [36]. They described a protocol where the EHS cells were propagated by
transplantation in mice and basement membrane components were extracted from the
tumor mass by breaking up protein–protein bonds. The subsequent viscous liquid solidi-
fied into a hydrogel at 37 °C and was later commercialized under the name Matrigel [37–
40]. Nowadays, different BME formulations (e.g., growth factor reduced or collagen type
IV enriched) are commercially available from various manufacturers (e.g., Corning Mat-
rigel or Cultrex BME). Exact production methods are proprietary information and could
therefore differ from previously described protocols.
BME is typically used as an in vitro replacement of the ECM, as it creates a bioactive
and biodegradable 3D environment for the cells. Commercially available BMEs are ready-
to-use formulations as no additional chemicals are required for solidification of the pre-
gel solutions. The non-cytotoxic viscous pre-gel solution remains a liquid at 4 °C and so-
lidifies into a relatively soft hydrogel at 37 °C. Moreover, the optical clarity of the extracts
allows for day-to-day monitoring. Therefore, BMEs have long been the golden standard
for many different types of in vitro assays, such as angiogenesis assays [41,42], (tumor)
cell migration assays [43] or for maintaining (induced) pluripotent stem cells undifferen-
tiated [19,44,45] (see Kleinman et al. [46] and Benton et al. [47] for brief overviews on these
applications). BME is used for the expansion of organoids for similar reasons. The bioac-
tive components allow the epithelial cells to self-organize into their typical organoid struc-
tures.
However, there are also disadvantages of the use of BME for the culture of organoids.
The exact composition is poorly defined and large batch-to-batch differences have been
reported [38,44,48]. BME also keeps cells in an undifferentiated and proliferative state
[19,39]. This could also hamper the differentiation of cholangiocyte organoids towards
Figure 2.
Cholangiocyte organoids can repair deficits in the biliary epithelium. This treatment can
restore adequate drainage of bile and prevent build-up of toxic bile inside the liver. Cholangiocyte
organoids can be infused into the biliary tree to repair the damaged biliary tree.
4. Basement Membrane Extract as Culture Substrates
The clinical application of cholangiocyte organoids is currently limited by the use of
non-GMP (good manufacturing practice)-compliant basement membrane extracts (BMEs)
in which organoids are typically cultured. BME is a complex mixture of extracellular matrix
(ECM) components derived from the tumor mass produced by Englebreth-Holm-Swarm
(EHS) mouse cells. These cells produce an abundance of basement membrane components,
which can be extracted and processed into hydrogels [
36
,
37
]. The main constituents of BME
are laminin-111, collagen type IV and enactin [
37
–
39
]. Other components include a myriad
of other bioactive ECM components and growth factors [
39
]. The extraction of these com-
ponents and subsequent reconstitution was first described in 1986 by Kleinman et al. [
36
].
They described a protocol where the EHS cells were propagated by transplantation in mice
and basement membrane components were extracted from the tumor mass by breaking up
protein–protein bonds. The subsequent viscous liquid solidified into a hydrogel at 37
◦
C
and was later commercialized under the name Matrigel [
37
–
40
]. Nowadays, different BME
formulations (e.g., growth factor reduced or collagen type IV enriched) are commercially
available from various manufacturers (e.g., Corning Matrigel or Cultrex BME). Exact pro-
duction methods are proprietary information and could therefore differ from previously
described protocols.
BME is typically used as an
in vitro
replacement of the ECM, as it creates a bioactive
and biodegradable 3D environment for the cells. Commercially available BMEs are ready-
to-use formulations as no additional chemicals are required for solidification of the pre-gel
solutions. The non-cytotoxic viscous pre-gel solution remains a liquid at 4
◦
C and solidifies
into a relatively soft hydrogel at 37
◦
C. Moreover, the optical clarity of the extracts allows
for day-to-day monitoring. Therefore, BMEs have long been the golden standard for many
different types of
in vitro
assays, such as angiogenesis assays [
41
,
42
], (tumor) cell migration
assays [
43
] or for maintaining (induced) pluripotent stem cells undifferentiated [
19
,
44
,
45
]
(see Kleinman et al. [
46
] and Benton et al. [
47
] for brief overviews on these applications).
BME is used for the expansion of organoids for similar reasons. The bioactive components
allow the epithelial cells to self-organize into their typical organoid structures.
However, there are also disadvantages of the use of BME for the culture of organoids.
The exact composition is poorly defined and large batch-to-batch differences have been re-
ported [
38
,
44
,
48
]. BME also keeps cells in an undifferentiated and proliferative
state [19,39].
This could also hamper the differentiation of cholangiocyte organoids towards mature
hepatocytes. Moreover, the constituents are not specific for liver tissue. Under normal
circumstances, the main constituent of BME (laminin-111) is not found in healthy adult liver
Bioengineering 2022,9, 110 5 of 17
parenchymal regions [
49
,
50
]. However, in situations where proliferating cells are required,
such as during embryonic development or regeneration after damage, laminin-111 can be
found here [
49
,
51
–
53
]. Evidence suggests that the presence of laminin can maintain the
stemness of certain cells and inhibit the differentiation of hepatic progenitor cells towards
mature hepatocytes [52,54,55].
5. Tissue-Specific Alternative Culture Substrates
The use of a culture substrate capable of closely mimicking the native liver ECM
could drive cells towards mature and functional cholangiocyte and/or improve differ-
entiation of ICO towards hepatocyte-like cells [
44
,
56
,
57
]. Tissue-specific microenviron-
ments can be built using liver ECM components and incorporation of cell signaling moi-
eties (e.g., growth factors). Gradients of growth factors play an important role during
embryonic development but are also important for maintaining tissue homeostasis or
are involved in tissue repair [
53
,
56
,
58
]. Spatiotemporal deposition of vascular endothe-
lial growth factor (VEGF) through the ECM, for example, guides vascular sprouting
during angio(neo)genesis [59–62].
Growth factors, such as endothelial growth factor (EGF)
or hepatocyte growth factor (HGF), are known to maintain the hepatocyte phenotype in cells
and are involved in the differentiation of stem cells towards hepatocyte-like cells [
63
,
64
]. In
addition, biophysical components (e.g., stiffness or elasticity) can influence the behavior
of cells through complex mechanotransduction pathways [
65
–
67
]. This has been shown
in vitro
with the differentiation of mesenchymal stromal cells, which can be directed by
altering matrix elasticity [
67
,
68
]. Growing hepatocytes
in vitro
on rigid matrices decreases
their ability to maintain a hepatocyte phenotype [
57
,
69
–
71
]. Similarly, an increase in liver
ECM stiffness is associated with a reduction in hepatocyte functionality and can lead to
liver fibrosis [
49
–
51
,
72
]. This shows the importance of having a culture substrate capable of
mimicking the native ECM.
Alternative culture substrates have to meet additional requirements. They must,
for example, allow for the expansion of the organoids in a GMP-compliant environment.
Organoids self-organize into 3D structures through complex cell–cell and cell–matrix
interactions. Therefore, the alternative substrate should also allow for the expansion of the
organoids by being capable of either deformation or site-specific degradation. Moreover,
the growth of cells also implies that the mass-transfer of oxygen, nutrients and metabolic
waste products through the substrate is required in order to maintain cell viability while
cell numbers are increasing.
5.1. Hydrogels as an ECM Mimic
Hydrogels are a promising class of materials for use as
in vitro
culture substrates [
73
].
Their polymeric networks are capable of maintaining relatively large amounts of water and
can form hydrated environments for cells
in vitro
. These networks are typically porous
and allow for mass-transfer to a certain degree [
73
]. Hydrogels have found widespread
use in biomedical applications such as contact lenses, drug delivery systems and wound
dressings [
74
]. The polymer backbones of hydrogels are typically derived from natural
or synthetic sources. Examples of natural polymers used for creating hydrogels are al-
ginate, fibrin, chitosan, cellulose and collagen [
73
,
75
,
76
]. Poly(acrylic acid), poly(vinyl
alcohol) and poly(ethylene glycol) (PEG) are examples of synthetic polymers [
74
,
76
]. These
polymers often require chemical modifications to create cross-linkable and/or degradable
networks [73].
Synthetic hydrogels are generally well defined and can be modified with relative ease.
This allows for the adjustment of, for example, the number of physical crosslinking sites,
which can alter the stiffness of the hydrogel. However, synthetic hydrogels often lack the
complexity of the native ECM in terms of mixtures of different bioactive ECM molecules
or degradation sites for cells [
77
]. PEG-based hydrogels were tested for expansion and
differentiation of intestinal organoids, but required incorporation of ECM components (e.g.,
fibronectin or laminin), cell adhesion motifs (RGD) or matrix metalloproteinase-degradable
Bioengineering 2022,9, 110 6 of 17
sites [
77
,
78
]. Nonetheless, Gjorevski et al. showed that relatively stiff hydrogels (~1.3 kPa)
increased the proliferation of these organoids, whereas differentiation to hepatocyte-like
cells was more optimal in a softer hydrogel [
78
]. Similar synthetic hydrogels also support
expansion and differentiation of human ICO, but still required the addition of ECM compo-
nents or cell-adhesion motifs [
79
]. Ye et al. used a hydrogel based on polyisocyanopeptides
(PIC) supplemented with laminin-111 and showed that ICO have similar proliferation rates
when compared to ICO grown in BME [
80
]. Nanocellulose hydrogels appeared to promote
differentiation towards hepatocytes [
81
]. However, it remains elusive whether these hydro-
gels can fully replace BME, as initiation of ICO in these hydrogels was not tested. Moreover,
nanocellulose hydrogels also required supplementation with ECM-components and it was
suggested by authors that further optimization regarding addition of ECM-components
could enhance ICO growth [81].
In short, creating a tissue-specific microenvironment capable of mimicking the native
ECM using synthetic hydrogels requires extensive experience in biochemistry and bioengi-
neering. Fine-tuning an optimal tissue-specific culture substrate is practically not possible
without the use of high-throughput screening methods [
82
]. However, even when using
high throughput screening methods, combining and testing multiple ECM components
is challenging and time consuming. This problem of recreating tissue mimicking culture
substrates can also be circumvented using extracts derived from decellularized healthy
tissues [83].
Liver decellularization procedures have been described for livers from different ani-
mals, such as rodents [
84
–
87
] and pigs [
58
,
88
–
94
], but also for whole human livers [
94
–
96
].
These protocols aim to remove all cellular components from liver tissue while retaining
the architecture of the ECM, including the highly tissue-specific spatiotemporal deposition
of the ECM components [
97
–
99
]. Subsequently, the ECM components can be extracted by
solubilizing the matrix using the enzyme pepsin in an acidic environment [
100
–
102
]. The
resulting extracts consist of bioactive and biodegradable ECM components, which can form
collagen-based hydrogels without the need for complex chemical modifications [103].
5.2. ECM-Based Hydrogels
Similar protocols exist for the extraction of collagen from collagen-rich tissues, such as
rat-tail tendon, fish scales or skin tissue [
104
], for biomedical applications (see [
103
] for a
comprehensive review on clinical applications of collagen-based materials). The pepsin
cleaves the non-helical telopeptide regions of collagen fibers but does not affect the helical
parts of the collagen. Subsequently, the helical parts are released by the enzyme, which can
reassemble themselves in long collagen fibers after the pH is normalized to
7.4 [103,105,106].
Similar gelation occurs after pH of the pepsin solubilized ECM is normalized to 7.4. The
viscous pre-gel solution self assembles into a collagen-based hydrogel, which contain nu-
merous ECM components [
83
,
107
]. The presence of ECM molecules influence the gelation
kinetics (e.g., crosslinking of fibril formation) of the ECM hydrogels [
108
] and creates
complex hydrogels with varying mechanical characteristics.
ECM based hydrogels are derived from biological sources and can, therefore, be
subject to biological variations. This can be mitigated by creating large batches of ECM
extracts from decellularized tissues. ECM extracts are also less well suited for studying the
effects of certain biochemical or biophysical components. The hydrogels are collagen-based
and altering the concentration also alters the biophysical characteristics and vice versa.
This could be resolved by incorporating the ECM extracts into a synthetic hydrogel. This
hybrid hydrogel could allow for alterations in biophysical characteristics (e.g., stiffness)
without altering the presence of biological components. Skardal et al. used solubilized liver
ECM to decorate simplistic PEG hydrogels [109].
5.3. Applications of Liver ECM Extracts
Liver ECM-derived extracts have been investigated for improving
in vitro
hepa-
tocyte cultures by using them as a supplement for culture medium [
110
], creating 2D
Bioengineering 2022,9, 110 7 of 17
coatings [
107
,
111
,
112
] of cell culture plastics or by creating bioactive 3D environments
for hepatocytes [
71
,
100
,
107
,
111
–
114
]. Further applications of liver ECM extracts include
the culture of other liver cells, such as hepatic stellate cells or liver sinusoidal endothe-
lial cells [
88
,
115
], or to improve the differentiation capacity of adipose derived stromal
cells [
107
] or pluripotent stem cells towards hepatocytes [
116
–
118
]. The liver ECM extracts
are also an attractive alternative culture substrate for the culture of cholangiocyte organoids.
Giobbe et al. showed that different endodermal organoids (including ICO) can be cultured
in non-tissue-specific ECM-derived hydrogels derived from decellularized porcine small
intestinal submucosa (SIS) [
119
]. SIS mainly consist mostly of a mesh of collagen fibers
and contains few other proteins [
120
]. Therefore, it might not represent a tissue-specific
ECM environment for the ICO. Liver ECM extracts are a favorable alternative, since they
are tissue-specific and could be of clinical relevance. The use of liver ECM extracts could
unlock the full clinical potential of the cholangiocyte organoids.
6. Finding a Suitable Source of Liver ECM
In theory, the decellularization procedure removes all cellular components (including
immunogenic proteins and DNA) from liver tissue. ECM components are also highly
preserved between species, allowing for the use of animal liver-derived extracts for clinical
applications in humans. However, retention of species-specific differences in liver ECM
extracts have previously been reported by Loneker et al. [
110
]. These differences can
partially be explained by biological variances between species, but also depend on tissue
processing (e.g., decellularization and enzymatic digestion). Detergents can, for example,
have detrimental effects on ECM components. We previously showed that
triton-X-100 +
Sodium dodecyl sulfate removes more collagen and sGAG from porcine liver ECM than
when only triton-X-100 was used for decellularization [
94
]. Similar effects have also been
reported by others who compared different treatment methods [
100
,
121
–
123
]. Decellular-
ization of human livers required more detergent and longer exposure times compared to
decellularization of similar sized porcine livers [
94
]. Subsequently, this could create larger
differences between human and porcine livers. Therefore, it is important to consider the
source of liver tissue.
Decellularized human livers are a promising allogeneic source of scaffolds for tissue
engineering applications and for preparation of liver ECM extracts. However, healthy
livers are relatively scarce. Human research livers (donor livers deemed unsuitable for
transplantation) were used in our previous studies. Further improvements in the field of
organ preservation and development of ex vivo repair strategies could in the future allow
for the safe transplantation of these research livers [
12
]. Thereby, the number of available
livers for research purposes will likely further diminish. Cadaveric livers could be an
alternative source of healthy human livers. One of the disadvantages of the use of human
livers is the relative old age and subsequent age-related changes of the
ECM [124–126].
With increased age comes increased stiffness and decreased elasticity due to scar tissue
formation and/or non-enzymatic crosslinking of the ECM [
127
]. The latter is caused by the
age-related accumulation of advanced glycation end-products (AGE) attached to the ECM.
This accumulation of AGE is influenced by different factors, such as dietary habits [
128
], and
can increase the aforementioned cross-links. These changes in ECM can prevent enzymatic
digestion of the ECM [
113
,
125
,
129
,
130
]. Subsequently, this can also lead to relatively large
differences between human livers [124].
Animal livers of similar age can be obtained with relative ease in a standardized
manner, thereby limiting the effect of age-related biological variances of the ECM. Small
animal livers (e.g., mice, rat or ferret) are typically well suited for small-scale recellular-
ization experiments. However, generating significant amounts of ECM extracts would
require sacrificing many animals. Porcine livers, on the other hand, are comparable in
size and weight to human livers. They are therefore promising alternatives for creating
tissue-engineering scaffolds, but also yield more liver ECM extracts per liver. The anatomy
of porcine livers is not similar to the anatomy of human livers. Porcine livers have
2–7 lobes,
Bioengineering 2022,9, 110 8 of 17
depending on breed [
131
]. Furthermore, the hepatic lobules of the porcine livers are sep-
arated by a clearly visible septa made of collagen-rich tissue (see Figure 3). These septa
resemble fibrotic human livers and are not present in human livers under normal healthy
circumstances [
72
,
131
]. The presence of these collagen-rich septa could shift the relative
distribution of ECM components present in the liver ECM extracts and this could influence
the behavior of primary human hepatocytes or stellate cells [124].
Bioengineering 2022, 9, x FOR PEER REVIEW 8 of 17
sacrificing many animals. Porcine livers, on the other hand, are comparable in size and
weight to human livers. They are therefore promising alternatives for creating tissue-en-
gineering scaffolds, but also yield more liver ECM extracts per liver. The anatomy of por-
cine livers is not similar to the anatomy of human livers. Porcine livers have 2–7 lobes,
depending on breed [131]. Furthermore, the hepatic lobules of the porcine livers are sep-
arated by a clearly visible septa made of collagen-rich tissue (see Figure 3). These septa
resemble fibrotic human livers and are not present in human livers under normal healthy
circumstances [72,131]. The presence of these collagen-rich septa could shift the relative
distribution of ECM components present in the liver ECM extracts and this could influ-
ence the behavior of primary human hepatocytes or stellate cells [124].
Figure 3. Porcine livers differ from human livers from an anatomical point of view. Porcine livers
have multiple lobes, but also contain septa (indicated by black arrows). These septa are not visible
in human livers. The scale bars represent 500 µm.
Biosafety Concerns of Using Decellularized Liver Tissue
The use of decellularized tissues in clinical settings is associated with biosafety con-
cerns. Inadequate processing of decellularized tissue can have detrimental effects on the
scaffold and cause adverse clinical outcomes, such as rapid degradation, loss of mechan-
ical integrity and/or inadequate tissue remodeling [132–134]. The presence of cellular rem-
nants, especially xenogeneic components such as α-gal (galactose-α-1,3-galactose) or
Figure 3.
Porcine livers differ from human livers from an anatomical point of view. Porcine livers
have multiple lobes, but also contain septa (indicated by black arrows). These septa are not visible in
human livers. The scale bars represent 500 µm.
Biosafety Concerns of Using Decellularized Liver Tissue
The use of decellularized tissues in clinical settings is associated with biosafety con-
cerns. Inadequate processing of decellularized tissue can have detrimental effects on the
scaffold and cause adverse clinical outcomes, such as rapid degradation, loss of mechan-
ical integrity and/or inadequate tissue remodeling [
132
–
134
]. The presence of cellular
remnants, especially xenogeneic components such as
α
-gal (galactose-
α
-1,3-galactose) or
MHC proteins [
135
,
136
], can influence the host response to decellularized ECM or solubi-
lized ECM (see [
137
,
138
] for comprehensive reviews on host response to animal-derived
decellularized ECM).
Bioengineering 2022,9, 110 9 of 17
Transmission of zoonotic diseases is another concern of using animal tissue-derived
materials for clinical applications. Porcine endogenous retrovirus (PERV) is an example of
a virus which could be transmitted [
139
]. Certain PERV subtypes can infect oversimplified
human cell cultures
in vitro
, but so far, there are no reports of humans which have been in-
fected with PERV after long-term exposure to pigs/porcine meat (e.g., farmers or butchers)
or patient who received porcine corneas or islets of Langerhans [
139
,
140
]. Moreover, others
have shown complete removal of detectable PERV provirus after complete decellularization
of porcine tissue [
140
,
141
]. Of note, allogeneic liver transplantation is not without risk of
transmitting human viruses [
1
]. Therefore, it is paramount that the decellularized livers
and subsequent extracts are screened for contaminants, immunogenic components and
(pro)viruses in order to mitigate these biosafety concerns.
7. Unlocking the Future Clinical Potential of Cholangiocyte Organoids
Tissue-specific ECM-based hydrogels are attractive culture substrates which can po-
tentially unlock the full clinical potential of cholangiocyte organoids. This potential is
not limited to use of the organoid-derived cells in cell therapy in ex vivo organ repair
strategies. Patient-derived cholangiocyte organoids can retain patient characteristics and
organoids cultures have been established for various hepatobiliary diseases, including
alpha1antitrypsin deficiency [
21
], cystic fibrosis [
27
], Alagille syndrome [
21
], primary
sclerosing cholangitis [
142
] and primary liver cancer [
143
,
144
]. Therefore, (personalized)
in vitro
disease models are an obvious application of the organoids (a comprehensive
review on the use of hepatobiliary organoids for disease modeling is published by Nucifero
and colleagues [
145
]). Cell–cell and cell–matrix interactions play important role during
the development of hepatobiliary diseases, as the onset is often associated with significant
changes in the ECM [
144
]. Decellularized liver tissue and liver ECM extracts can play im-
portant roles in creating these disease models. Moreover, the biological variances could also
be embraced as a means to study the effect of different environments (e.g., relatively young
ECM versus relatively old ECM [
125
,
127
]) on the behavior of cells or on the development
of certain diseases [
144
]. Ultimately, patient-derived organoids cultured in tissue-specific
matrices could lead to improved treatment strategies for hepatobiliary diseases.
7.1. Tissue Engineering the Biliary Tree
Tissue-engineered functional liver constructs have the potential of bridging the gap
between the demand and supply of donor livers of adequate livers [
146
–
148
]. Creating
liver constructs
in vitro
requires scaffolds that are capable of performing similar roles as
the native liver ECM. Current production techniques for alternative ECM or synthetic
supporting structures, such as 3D bio-printing, can recreate small constructs mimicking
the liver architecture with high fidelity, but producing clinically relevant sized scaffolds
is still challenging [
149
,
150
]. Use of the native liver ECM is an attractive alternative
to de novo creation of scaffolds with synthetic materials [
97
,
147
,
148
]. In recent years,
recellularization with primary hepatocytes or hepatocytes derived from various stem
cell sources have been investigated in effort to restore functionality of the hepatocyte
compartment [71,151–153].
Simultaneously, a lot of effort has been invested in repopulation
of the vasculature network [
95
,
148
]. However, repopulation of the biliary compartment
has not yet been extensively studied, even though biliary epithelium is essential for proper
functioning of the liver [9,154].
Cholangiocyte organoid-derived cells are a promising source of cells for repopulation
of the biliary tree. We recently showed that cells derived from ECO and BCO were capable
or repopulating small discs (Ø3 mm) of decellularized extrahepatic bile duct (EBD) tis-
sue [
30
,
155
]. They self-organized into polarized monolayers resembling biliary epithelium
(Figure 4). Moreover, the repopulated scaffold showed increased trans epithelial electrical
resistance and cholangiocyte specific ion-channel activity could be measured. This could
be translated into transplantable EBD constructs, but the organoids could also be used to
repopulate the biliary tree of decellularized liver ECM (Figure 4) [
25
,
156
]. However, also
Bioengineering 2022,9, 110 10 of 17
here the use of BME as a culture substrate hampers the clinical applications of these lab-
grown biliary structures. Clinical grade alternatives are required for the
in vitro
expansion
of organoids, before repopulated biliary trees can be used in vivo.
Bioengineering 2022, 9, x FOR PEER REVIEW 10 of 17
epithelium (Figure 4). Moreover, the repopulated scaffold showed increased trans epithe-
lial electrical resistance and cholangiocyte specific ion-channel activity could be meas-
ured. This could be translated into transplantable EBD constructs, but the organoids could
also be used to repopulate the biliary tree of decellularized liver ECM (Figure 4) [25,156].
However, also here the use of BME as a culture substrate hampers the clinical applications
of these lab-grown biliary structures. Clinical grade alternatives are required for the in
vitro expansion of organoids, before repopulated biliary trees can be used in vivo.
Together with more liver-specific cell types, such as hepatic stellate cells and Kupffer
cells, a transplantable tissue-engineered liver construct can be made. Decellularized liver
tissue can be applied in different stages and in different forms for creating tissue-engi-
neered liver constructs in vitro. Solubilized liver ECM hydrogel can, for example, be used
for the clinically relevant expansion of cholangiocyte organoids in vitro, while decellular-
ized livers can be used as a bioactive, biodegradable and inductive scaffold for liver tissue
engineering purposes in vitro and in vivo.
Figure 4. Potential future tissue engineering applications of human cholangiocyte organoids. (A)
Cholangiocyte organoids can also be used to repopulate extrahepatic bile duct (EBD) scaffolds for
ductal tissue engineering purposes. Subsequently, these engineered ductal scaffolds can be used to
replace damaged tissue. (B) An example of a strategy for recellularization of decellularized liver
ECM. Cholangiocyte organoids can be used to repopulate the entire biliary tree. Endothelial cells
can recellularize the vasculature of the liver and ICO differentiated towards hepatocyte-like cells
can be used to repopulate the hepatocyte compartment. These different types of cells could restore
functionality of the liver.
Figure 4.
Potential future tissue engineering applications of human cholangiocyte organoids. (
A
)
Cholangiocyte organoids can also be used to repopulate extrahepatic bile duct (EBD) scaffolds for
ductal tissue engineering purposes. Subsequently, these engineered ductal scaffolds can be used to
replace damaged tissue. (
B
) An example of a strategy for recellularization of decellularized liver
ECM. Cholangiocyte organoids can be used to repopulate the entire biliary tree. Endothelial cells
can recellularize the vasculature of the liver and ICO differentiated towards hepatocyte-like cells
can be used to repopulate the hepatocyte compartment. These different types of cells could restore
functionality of the liver.
Together with more liver-specific cell types, such as hepatic stellate cells and Kupffer
cells, a transplantable tissue-engineered liver construct can be made. Decellularized liver
tissue can be applied in different stages and in different forms for creating tissue-engineered
liver constructs
in vitro
. Solubilized liver ECM hydrogel can, for example, be used for
the clinically relevant expansion of cholangiocyte organoids
in vitro
, while decellularized
livers can be used as a bioactive, biodegradable and inductive scaffold for liver tissue
engineering purposes in vitro and in vivo.
7.2. Summary
Mouse tumor-derived BMEs are commercially available and easy-to-use formulations
which allow for organoid growth and expansion. However, these one-size-fits-all BME
Bioengineering 2022,9, 110 11 of 17
formulations limit the direct clinical application of cholangiocyte organoids for cell therapy
or tissue engineering applications. In addition, BME lacks essential tissue-specific ECM
components and is known to keep cells in a high proliferative undifferentiated state. There
is a clear need for a tissue-specific alternative which can be produced according to GMP
guidelines. This alternative must also allow for the culture and large-scale expansion of
these organoids. Hydrogels derived from healthy decellularized and solubilized liver ECM
are promising alternative culture substrates for the large-scale expansion of cholangiocyte
organoids and could unlock the enormous clinical potential of the organoids.
Author Contributions:
Writing—original draft preparation, J.W.; writing—review and editing,
L.J.W.v.d.L., J.d.J. and M.M.A.V.; visualization, J.W.; funding acquisition, L.J.W.v.d.L., J.d.J. and
M.M.A.V. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by Medical Delta program grant, Regenerative Medicine 4D;
TKI-LSH grant, project number EMC-LSH19002 and NVGE Gastrostart fund, project number 2019-06.
Conflicts of Interest: The authors declare no conflict of interest.
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