JUST, Vol. V, No. 1, 2017
Trent University
In Vitro Organogenesis of the Liver: Current
Progress and Future Applications
Eliza McColl
Abstract
Tissue engineering, the process of growing cells on a 3D scaffold to form organ-like structures, represents a
growing field of in vitro developmental biology. Since receiving its name in 1987, the field of tissue engineering
has evolved considerably from being used to grow simple tissues such as skin and cartilage to generating 3D,
semi-functional precursors to organs such as the pancreas, kidney, heart, and liver. However, the generation of
these more complex organs requires the ability to generate extensive vascularization and condense complex
cell mixtures, both of which have thus far provided a major barrier in culturing full-sized, functional organs. In
order to advance to culturing full-sized, functional livers in vitro, current research is striving to optimize the
cell compositions, culturing conditions, and 3D scaffolds used for in vitro liver organogenesis. This review
outlines recent studies that have made important advances regarding protocols used to generate 3D-culture liver
organoids as well as their potential applications in health care.
Keywords
Tissue Engineering — Developmental Biology — Health Sciences
Champlain College
1. Introduction
1.1 What is Tissue Engineering?
The term “tissue engineering” refers to the use of living cells,
biocompatible materials, and other factors to create tissue-like
structures in vitro (Berthiaume et al. 2011). The process of tissue engineering typically involves using 3D scaffolds as structural support to encourage embryonic stem cells, adult stem
cells, or mature cells to condense and develop based on natural
cell affinities to form new tissue (Cortesini 2005; Stamatialis
et al. 2008). The 3D scaffolds are often biological in origin,
such as protein-based materials and polysaccharide-based materials that have been cross-linked with synthetic agents to
prevent the scaffold from degrading over time (Berthiaume
et al. 2011). Solely synthetic scaffolds are often avoided
due to their low biocompatibility. These matrices are seeded
with stem cells that are then induced to differentiate using
cell line-specific chemicals that mimic endogenous signals
required for proliferation. This allows the cells to proliferate
into different cell types to generate 3D tissue and organ-like
structures in vitro. The ultimate goal of in vitro tissue generation is to generate tissue or organs that can be implanted into
the human body or used to repair failing organs (Berthiaume
et al. 2011).
The first successes in the field of tissue engineering were
with regards to skin and cartilage generation; generating these
tissues was relatively simple (compared to engineering other
organs) because it only required combining cells and 3D matrices, as these tissues don’t require extensive vascularization to
perform their endogenous functions (Berthiaume et al. 2011).
The true challenge in tissue engineering arises when this technology is applied in an attempt to generate more complex
organs that require vasculature and diverse cell compositions
to function properly. For this reason, the only whole tissueengineered organ to have been successfully grown in vitro at
this point is a trachea. However, substantial advances have
been made with regards to other organs such as the pancreas,
kidney, heart, spinal cord, and the liver.
1.2 The Liver: An Important Target for Culturing
The main targets of modern tissue engineering are organs
that are commonly prone to injury, disease, and degeneration
(Berthiaume et al. 2011). For this reason, the liver is a
prime candidate for in vitro organogenesis. Currently, the
only successful treatment for patients with end stage liver
disease is liver transplantation; however, 20% of end stage
liver failure patients die on the waiting list for a transplant
due to a shortage of donors, and many patients are not eligible
for transplantation in the first place due to the severity of
their illness (Berthiaume et al. 2011; Mazza et al. 2015).
Additionally, the total number of deaths worldwide caused by
cirrhosis and liver cancer has been increasing by 50 million
per year since 1990 (Mazza et al. 2015). Due to the increasing
number of deaths related to liver cirrhosis, cancer, and the
lack of viable transplantation options it is necessary for novel
treatments to be assessed.
Using tissue engineering to grow livers in vitro for the
purpose of transplantation represents only one of many applications of in vitro liver organogenesis. The ability to engineer
livers in vitro could also have applications such as providing
In Vitro Organogenesis of the Liver: Current Progress and Future Applications — 2/6
better models for studying human disease and testing novel
therapeutics. Since the liver plays a prominent role in metabolizing xenobiotics, livers cultured in vitro could not only
be used to test the response of liver tissue to novel therapeutics, but could also give an indication of the metabolism of a
given drug which would aid in determining proper therapeutic
doses during preclinical trials (Lancaster & Knoblich 2014).
However, only small, 3D liver precursors, termed “organoids”,
have successfully been cultured in vitro to date, with the first
liver organoid being successfully cultured only six years ago
(Lancaster & Knoblich 2014).
This review will outline some of the challenges currently
preventing full-sized liver culturing, how current research is
striving to overcome these challenges, and the potential applications that in vitro liver organogenesis could offer. Research
aiming to improve current liver organoid culturing methods,
inspire progression towards fully cultured livers in vitro, and
implicate the applications of these endeavours are novel fields
of developmental biology and thus represent an exciting current topic in biochemistry.
2. Current Research: Striving to
Overcome the Hurdles of Growing Full
Livers
While a full-sized liver has not yet been grown in vitro, many
studies have had success growing liver organoids or organ
buds, which are small-scale precursors of the liver grown in
vitro (Lancaster & Knoblich 2014). A major barrier faced
when attempting to progress from engineering liver organoids
to full-sized organs is the ability to expand 3D liver progenitors for long periods of time in vitro (Broutier et al. 2016).
For example, a previous study by Michalopolous et al. (2001)
found that when trying to culture hepatocytes, apoptosis occurred in 50% of the cultured cells after only five days. Even
more recently, 3D hepatocyte cultures have only been sustained for approximately one week which only allowed for a
10-fold expansion of the culture (Shan et al. 2013). Without
the capability to culture organ progenitors for long periods of
time, cells composing liver organoids do not have the opportunity to self-organize into functional components of the liver,
such as the hepatic ductal compartment, or the vascularization
required to support the liver’s growth and function (Broutier
et al. 2016). Therefore, culturing methods must be improved
in order to progress towards culturing entire functional livers
in vitro.
In an attempt to overcome the barrier of long-term liver
organoid culturing, Broutier et al. (2016) have developed a
novel culturing method for 3D liver organoids. This method
has increased the length of time that liver progenitors can
be cultured in vitro from a couple weeks at maximum to as
long as three months. This new culturing method involves
combining a scaffold called Matrigel, which has properties
similar to the extracellular matrix, with hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth
factor (EGF), and R-spondin-1 (Rspo1). HGF, FGF, EGF,
and Rspo1 are factors responsible for embryonic development, progenitor proliferation, and migration of liver cells.
It had previously been established that EGF, HGF, and dexamethasone aided in the maintenance of primary liver cells
in culture. The combination of these factors with FGF and
Rspo1 resulted in the improved culture medium proposed by
Broutier et al. (2016). When combined with the Matrigel
matrix, these additional factors contributed to the survival
and genetic stability of liver organoids over longer periods
of time (months) in vitro. This survival and stability allowed
single liver progenitors to give rise to up to 106 cells, allowing
sufficient time for the cells to self-organize into an epithelial
structure that resembled the hepatic ductal compartment as
well as compartments that partially resembled liver buds of
an early embryo.
Shifting liver organoid cultures to a differentiation medium
that promotes the expression of hepatocyte markers allowed
the cells composing the liver organoids to develop functional
hepatocyte characteristics (Broutier et al. 2016). The differentiation medium contained dexamethasone and bone morphogenetic protein (BMP), both of which promote hepatocyte
differentiation, as well as components that blocked Notch,
a factor that promotes ductal development. Blocking ductal
development was an important aspect as it promoted differentiation of the organoids cells into functional hepatocytes as
opposed to just ductal cells. The resulting differentiated cells
produced albumin or bile acid in vitro which indicated hepatocyte functionality. This advancement in the ability to culture
liver organoids for months as opposed to days offers great
potential for growing full, functional livers in vitro. However,
this procedure for long-term culturing can so far only be done
using the epithelial components of the liver tissue and has
not yet shown success with liver organoids that contain both
mesenchymal (multipotent cells) and epithelial cells, which
would be required to achieve the complexity of full livers.
The challenge faced by Broutier et al. (2016) when attempting to culture multiple cell types in unison is another
common barrier faced by in vitro liver engineering. For this
reason, many of the current examples of successful tissue
engineering are derived from only epithelial structures and
lack more complex structures such as vasculature (Takebe et
al. 2015). In vivo, the liver develops from a combination of
mesenchymal stem cells, undifferentiated vascular endothelial cells, and endoderm cells. In order to generate livers in
vitro that closely resemble and function like livers in vivo, a
protocol must be developed that allows liver organoids to be
composed from cultures containing multiple cell types.
Takebe et al. (2015) have developed a protocol, depicted
in Figure 1, in which mesenchymal stem cells are used to initiate condensation of heterotypic cell mixtures which allowed
them to generate vascularized and complex liver organoids.
This protocol eventually led to the first ever generation of
human-like liver organoids, as previous successful liver organoids
had only been grown from animal cells lines (Lancaster &
In Vitro Organogenesis of the Liver: Current Progress and Future Applications — 3/6
organoid generation of not only the liver, but multiple other
organs.
Figure 1. Schematic showing the mesenchymal-directed
condensation of multiple cell types to generate vascularized organ
buds of multiple organ types. Image courtesy of Takebe et al. (2015).
Knoblich 2014). Previous research had established that mesenchymal stem cells can enhance the contraction force of the
self-organization of cells (Sondergaard et al. 2010). Takebe
et al. (2015) implemented the use of the mesenchymal stem
cells to apply this knowledge to growing liver organoids. In
their initial research, they labelled human induced pluripotent
stem cell (iPSC)-derived hepatic endoderm cells and umbilical
cord-derived endothelial cells with two different fluorescent
markers, co-cultured them on a 3D matrix, and then used
fluorescent live imaging to observe any cell migration or condensation.
It was observed that in the absence of mesenchymal stem
cells, the co-cultures of the two different cell types failed to
condense into liver organ buds. Conversely, when mesenchymal stem cells were added to the co-culture of the endoderm
and endothelial cells, the mesenchymal stem cells appeared
to increase the contraction force to result in condensation
of the two cell lines. The live image cell tracking was also
used to observe that the condensed cells eventually developed
into vascularized liver organoids, as opposed to epithelialbased organoids. This phenomenon was observed in culture
medium similar to that designed by Broutier et al (2016) in
that it contained factors for hepatocyte development such as
dexamethasone and hepatocyte growth factor (HGF). This
protocol was also successful in developing complex organ
buds for the intestine, lung, kidney, heart, and brain, all of
which are pictured in Figure 2. The success of Takebe et al.
(2015) in generating complex, vascularized liver organoids
demonstrates the potential role of mesenchymal stem cells in
guiding the condensation of multiple cell types for complex
A third ongoing challenge in the field of in vitro liver
engineering is the optimization of an ideal 3D scaffold. In
order for a liver to be grown to full size in vitro and maintain functionality, the 3D matrix on which it is grown must
support the growth and differentiation of the cells composing
the organoid (Mazza et al. 2015). Additionally, as previously
mentioned, growing livers with sufficient vascularization for
proper functioning and survival has also been a challenge.
This vascularization would be required in order to supply oxygen and nutrients to the cultured tissue for long-term survival
(Cortesini 2005). Thus, if the matrix used to grow the liver
could somehow already exhibit this vascularization, it would
greatly enhance the ease with which functioning livers could
be grown in vitro. In an attempt to achieve an ideal 3D scaffold, Mazza et al. (2015) proposed using decellularized liver
tissue as a 3D scaffold. This technique had previously been
shown to promote successful growth of multiple liver cells
types while retaining their functionality. For example, Uygun
et al. (2010) were the first to successfully decellularize rat
livers and use them to create tissue-engineered liver grafts
with functioning hepatocytes. However, all previous research
using this technique had been done using animal liver tissue
and until recently it was not known whether this technique
could be applied to human liver tissue.
Mazza et al. (2015) used healthy human livers deemed
ineligible for transplantation and decellularized them using a
retrograde perfusion protocol. The purpose of this protocol
was to remove immunogenic cellular materials from the livers
while maintaining the 3D shape, essential matrix proteins, and
vascularization of the organ so that it could be used to generate
fresh tissue in vitro. The perfusion protocol involved flushing
the livers (either a single lobe or a whole liver) with solutions containing varying concentrations of detergents such as
sodium dodecyl sulfate (SDS) and Triton X100. Throughout
the perfusion, the flow rate of the decellularization solutions
was varied; a slow flow rate was used to begin the decellularization and then was rapidly increased before being stabilized
for the remainder of the decellularization. The decellularized livers (shown in Figure 3) were then examined using
immunostaining analysis, as well as techniques that quantified
DNA, collagen, and elastin present in the matrices to determine the success of the perfusion. Mazza et al. (2015) were
then able to successfully re-engraft three different human liver
cell types (LX2, HepG2, and Sk-Hep-1 cells) onto cubes of
the decellularized matrix by using a syringe to deposit the
cells onto the cubes. Using tissue staining techniques, successful migration and cellular proliferation of each of the cell
types on the matrix was observed. This success represents the
potential for decellularized tissue to provide an optimal 3D
basis for new full-sized, vascularized, functional liver formation, specifically in humans. The donation of the livers to be
decellularized could come from organs that are deemed unfit
for transplantation, which thus would not deprive a transplant
In Vitro Organogenesis of the Liver: Current Progress and Future Applications — 4/6
Figure 2. The protocol derived by Takebe et al. (2015) in which mesenchymal-directed condensation of multiple cell types is used for organoid
formation resulted in successful generation of organoids of multiple tissue types. Image courtesy of Takebe et al. (2015).
cantly improve our knowledge of human disease development
and the response of tissue to novel therapeutics. The models
currently used to study these processes include 2D cell culture methods, animal models, or cadaver tissues are used to
study these processes; however, all three of these methods
have significant shortcomings. For example, 2D cultures fail
to take into account the microenvironment of 3D tissues in
vivo and thus have limited applicability to in vivo conditions
(Skardal et al. 2015). Similarly, animal models don’t always
resemble human tissue types and can yield results that don’t
translate to human tissue (Elliott & Yuan 2010). While human
cadaver tissues may offer the most accurate depiction of live
human tissue, problems occur due to a lack of donor tissue and
difficulty associated with maintaining the viability of excised
tissues (Elliott & Yuan 2010). Therefore, there is a great need
for improved models to more effectively study human disease
and drug response.
Figure 3. Decellularized left lobe of a human liver. The perfusion
process results in the tissue taking on a translucent appearance as
the liver becomes increasingly decellularized. When lit up from
behind, the conserved vasculature of the decellularized matrix is
visible. Image courtesy of Mazza et al. (2015).
recipient of a viable organ. However, questions still remain
about optimal ways to recellularize the tissue to regenerate
the more complex areas of the liver including the hepatic
sinusoids and the portal triad vasculature.
3. Applications of In Vitro Organogenesis
of the Liver
The contributions of the research discussed above will hopefully aid in the ability to move toward growing full-sized,
functional livers in vitro, which would have multiple applications in the medical field. Three main applications will be
focused on: the generation of livers to be used for modelling
human disease, using these models to test the response of
liver tissue to drugs, and the potential for engineered livers
to be used for transplantation purposes. To begin, 3D in vitro
models of functional, viable livers have the potential to signifi-
The generation of viable, functional human liver tissue in
vitro could offer an invaluable model for studying liver disease
and drug response. Skardal et al. (2015) demonstrated the
ability to engineer a 3D liver-tumor hybrid organoid system
that could be used to study both tumor development and the
response of the tissue to chemotherapeutic treatment. In order
to achieve this, they generated 3D liver organoids composed
of both healthy liver cells and fluorescently labelled carcinoma
cells by co-culturing them in the presence of a 3D matrix in a
rotating wall vessel (RWV) bioreactor. The use of the RWV
allowed the cell cultures to be suspended in microgravity
which caused them to condense based on natural cell affinities
into liver organoids containing tumor foci representative of
metastatic colon carcinoma. The researchers were able to
study the development of the tumor foci within the healthy
liver tissue over time by imaging the developing liver-tumor
hybrid organoids with both light and fluorescence microscopy.
Figure 4 depicts the result of merging images of the growing
organoids taken using these two visualization techniques.
Imaging the organoids using these techniques gave the
researchers insight into how the tumors (composed of fluorescent carcinoma cells) formed within the healthy liver tissue over time. Upon confirming the viability of the resulting
organoids by measuring increases in their size and metabolism
In Vitro Organogenesis of the Liver: Current Progress and Future Applications — 5/6
Figure 4. Merged light microscopy and fluorescence microscopy images that show the development of tumor foci (red) within the liver organoids
over time during culturing. The transparent circles in the images are hyaluronic acid-coated microcarriers, which were used as the 3D matrix for
culturing. Image courtesy of Skardal et al. (2015).
during development, the researchers suggested that the model
could be used as a method for real-time tumor tracking. They
were also able to use immunohistochemical staining of multiple cell markers to determine that the phenotype of the cells
composing the organoids more closely resembled that of liver
cancer in vivo than those of typical 2D cultures. These results
support the potential for liver organoids to be used as more
representative model of human disease development.
In addition to exemplifying the potential of in vitro liver
engineering to provide a better model for disease development,
Skardal et al. (2015) were able to simultaneously demonstrate
the potential for this technology to be advantageous in testing therapeutics. Skardal et al. (2015) used their liver-tumor
organoids to test the specificity of a common chemotherapeutic agent, 5-fluorouracil (5-FU), in targeting the cancer cells
versus the surrounding healthy liver cells. By incubating the
organoids in 5-FU and counting how many of the cells that underwent apoptosis were cancer cells versus normal liver cells,
they were able to establish at what concentration 5-FU began
non-selectively targeting cancer cells and imposed damage
on healthy cells. Considering that the phenotype of the cells
composing the organoids was more representative of that of
an in vivo liver cancer system, these results are likely more
applicable to human disease and drug response in vivo. This
would therefore supply researchers with more accurate data
related to drug dosage for chemotherapies and how tissue may
react to new therapeutics.
Perhaps the most compelling application of in vitro liver
organogenesis is the potential to generate transplant organs.
As previously mentioned, the only current treatment option
for end stage liver disease is a liver transplant, but a shortage
of donors causes 20% of patients on the transplant wait list
to die before receiving a liver (Mazza et al. 2015). Thus,
the ability to transplant livers without the need for a donor
could alleviate the number of patients that die waiting for a
transplant. Current research is showing the potential of this
application by generating liver organoids and transplanting
them into animal models to see how they function and are
accepted in vivo. For example, Saito et al. (2011) successfully
grew liver organoids and transplanted them into mice. The
liver organoids were grown from a combination of immortalized mouse hepatocytes, hepatic stellate cells, and sinusoidal
endothelial cells on a 3D matrix in a radial flow bioreactor
(RFB). Within the RFB, culture medium containing the three
cell types was perfused over the 3D scaffold (AFS scaffold) to
produce viable liver organoids. The resulting organoids were
then transplanted into either a pocket under the renal capsule
of the kidney or the porta-hepatis region of the omentum in
mice; both locations were chosen based on the ease of the
operation required to perform the transplant. At either 4 or 8
weeks post-transplant, the mice were euthanized and the transplanted organoids were examined. Histological staining and
scanning electron microscopy (SEM) of the excised organoids
were used to determine that the transplanted organoids were
able to proliferate and survive in vivo post-transplantation.
Additionally, Saito et al. (2011) observed that the expression
of genes commonly transcribed in the liver (albumin, HNF-4α
and G6Pase) was higher in the organoids after the transplant
than during culturing. The results of this research indicate
the potential for engineered livers to be successfully transplanted into live patients to continue to develop and maintain
functionality. Not only would this reduce the wait time of
patients requiring a liver transplant, but it could also reduce
the risk of transplant rejection if the livers could be grown
using a patient’s own stem cells. Overall, this would lower the
number of patients that die from liver disease; however, this
technology would first need to be tested in human patients
as opposed to animal models to assess its true application in
health care.
4. Conclusion
In conclusion, while the current state of in vitro liver organogenesis represents significant advances in the field of tissue
engineering since the formation of more simple structures
such as skin or cartilage, various challenges still remain that
have thus far prevented the ability to culture a full-sized, functional liver in vitro. Current research in this field is attempting to optimize the cell types, culturing parameters, and 3D
matrices required to overcome these challenges. This is a
worthwhile pursuit, as the ability to generate a functioning
liver from a patient’s own stem cells in vitro could not only
be useful for transplantation purposes, but also for gaining a
better understanding of liver diseases and drug response of
tissues in vivo.
In Vitro Organogenesis of the Liver: Current Progress and Future Applications — 6/6
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