Published at : 18 Sep 2024
Volume : IJtech
Vol 15, No 5 (2024)
DOI : https://doi.org/10.14716/ijtech.v15i5.6088
Adrian Pragiwaksana | Stem cell and tissue engineering research cluster IMERI, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya no 6 Jakarta Pusat, 10430, Indonesia |
Muhammad Irsyad | 1. Department of Mechanical Engineering, Faculty of Engineering, Universitas Indonesia, Depok, Jawa Barat 16424, Indonesia 2. Medical Technology Cluster, cluster IMERI, Faculty of Medicine, Universit |
Muhammad Hanif Nadhif | 1. Medical Technology Cluster, cluster IMERI, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya no 6 Jakarta Pusat, 10430, Indonesia 2. Medical Physics Department, Faculty of Medicine, Univ |
Akhmadu Muradi | Division of Vascular and Endovascular Surgery, Department of Surgery, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya no 6 Jakarta Pusat, 10430, Indonesia |
Chyntia Olivia Maurine Jasirwan | Division of Hepatobiliary, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya no 6 Jakarta Pusat, 10430, Indonesia |
Vetnizah Juniantito | Division of Pathology, School of Veterinary Medicine and Biomedical Sciences, IPB University, Jl. Agatis, Bogor, Jawa Barat 16680, Indonesia |
Ridho Ardhi Syaiful | 1. Division of Digestive Surgery, Department of Surgery, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya no 6 Jakarta Pusat, 10430, Indonesia 2. Department of Surgery, Institute of Gastro |
Radiana Dhewayani Antarianto | 1. Stem cell and tissue engineering research cluster IMERI, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya no 6 Jakarta Pusat, 10430, Indonesia 2. Department of Histology, Faculty of Med |
In clinical terms, end-stage liver disease is a group of liver diseases that includes advanced liver disease, liver failure, and decompensated cirrhosis. Liver transplantation has been the most effective treatment for cirrhosis. The limited number of available and suitable living liver donors is a significant limitation in liver transplantation. Acute or chronic rejection could be the cause of liver transplant failure. To overcome rejection, usage of the long-term immunosuppressive drug is a standard post-transplant regimen. However, this therapy can increase the risk of severe viral or fungal infection and malignancy. Various attempts were made to address the liver transplant shortage. One of them is liver tissue engineering. This research was conducted with an artificial liver prototype of Stem Cell Hepatic Intuitive Apparatus (SHiNTA) with a perfusion bioreactor, whose manufacturing process is simple in the form of a liver microstructure consisting of differentiated hepatocytes from an hiPSCs from a modification of the Blackford protocol in a liver biologic scaffold. Liver biologic scaffolds were made from pieces of rabbit liver stored in the Stem Cell and Tissue Engineering (SCTE) laboratory, Fakultas Kedokteran Universitas Indonesia, by decellularization. This study aimed to develop the SHiNTA BALs to sustain the viability of hiPSC-derived hepatocytes in the artificial liver prototype. SHiNTA artificial liver prototype with a perfusion bioreactor connected to a perfusion pump with a specific perfusion rate of 2mL/min showed a higher cell count and confluence, with evenly distributed in the extracellular matrix than SHiNTA blood bag with orbital shaker group up to day 7. Furthermore, the SHiNTA artificial liver with perfusion bioreactor showed a positive signal of cell maturation in the scaffold (ASGPR, HNF4- , and CEBP- ) through immunofluorescence.
Bioreactor; hiPSCs; Histology; SHiNTA; Tissue engineering
End-stage liver disease is a term used to describe a
collection of liver conditions, including severe liver disease, liver failure,
and decompensated cirrhosis. In the USA, there are currently 11,514 people on
the liver transplant waiting list, and approximately 70.19 percent of them
received a transplant in 2017 (Kim et al., 2019). According to WHO data from 2012, the
mortality rate for liver cirrhosis in Indonesia is 52.7 (for males) and 16.6
(for women) per 100,000 deaths (Kim et al., 2016). Cirrhosis has been most successfully
treated through liver transplantation. A key barrier to liver transplantation
is the death of appropriate and available living liver donors. The failure of a
liver transplant may result from acute or chronic rejection. To prevent
rejection, the standard post-transplant procedure involves the use of long-term
immunosuppressive drugs. However, this therapy has been associated with an
increased risk of severe viral or fungal infections and malignancies. Various
approaches have been utilized to address the shortage of liver transplants. The
transplantation of hepatocyte cells as a therapeutic approach is limited by the
low engraftment rate of the cells, which in turn requires a large number of
hepatocyte cells (Alwahsh, Rashidi, and Hay, 2018). The present study reports that the
engrafted cells fail to differentiate into functional liver cells, leading to
the requirement of liver transplantation (Hamooda,
2016). Plasmapheresis,
hemodiafiltration, and artificial liver (AL) have been proposed as potential
alternatives to liver transplantation in the field of liver tissue engineering (Lee,
Kim, and Choi, 2015).
The field of
liver tissue engineering primarily focuses on four primary endeavors (Hosseini et al., 2019): 1. The development of complete, functional, and
implantable liver constructs; 2. The creation of bioartificial liver (BAL)
systems to support the lives of patients awaiting liver transplantation and in
vitro hepatocyte-based models; 3. The establishment of culture models for drug
metabolism and toxicity screening in drug discovery; 4. The provision of a
foundation for researchers studying liver regeneration, disease,
pathophysiology, and pharmacology. Like other types of tissue engineering,
liver tissue engineering requires three main components that are interconnected
and have an impact on one another: cells, scaffolds, and signaling molecules,
like growth factors and other active biomolecules. Complex tissue architecture
is created when cells derived from primary or stem cells are combined with
three-dimensional scaffolds. Scaffolds and signaling molecules work together to
provide structural, biochemical, and biomechanical support for cell and tissue
development (Sibuea, 2021). Recent progress in the
development of implantable engineered hepatic tissues has demonstrated
promising potential as a feasible alternative to overcome the constraints
associated with existing cell-based strategies. The limited engraftment of
cells and their short-term survival post-implantation are significant
challenges that remain to be addressed. Various methodologies have been
utilized to generate hepatic micro-tissues, including cell encapsulation, 3D
printing, microfluidic systems, and decellularization/recellularization
techniques (Heydari et al., 2020; Forbes and
Newsome, 2016).
The AL
technique integrates blood dialysis treatments with extracorporeal perfusion
system equipment, utilizing non-biological hemofiltration to effectively
cleanse the blood of individuals suffering from acute and chronic liver
failures. The primary function of AL is to facilitate liver transplantation by
eliminating metabolic waste and blood toxins while also preventing potential
damage to the brain and kidneys. The device's efficacy is limited to providing
temporary relief and stabilization of the patient's symptoms for a brief
duration. Therefore, liver transplantation continues to be imperative. A BAL,
specifically the hollow fiber bioreactor that involves the adherence of
hepatocytes within a cartridge, was applied to hollow fiber membranes that serve
as a scaffold for cell attachment and compartmentalization. The methodology
involves the integration of a synthetic liver with operational hepatocytes to
reconstruct or restore the metabolic function that has been compromised due to
liver cell loss. This enables partial replacement of the patient's hepatic
function during the period of awaiting liver transplantation (Vacanti and Kulig, 2014; Zhang et al., 2014).
Bioreactors are
utilized in tissue engineering for three primary purposes: 1.) Replicate the in
vivo state of cells in vitro, thereby facilitating the comprehension of normal
cellular and molecular physiology; 2.) Expand cells for potential clinical applications,
such as gene and cell therapies, or simulate a pathological state to study
pathophysiology; 3.) Employed to establish novel therapeutic targets and
evaluate potential new treatments in a more realistic setting than traditional
in vitro culture. Success in this area would also reduce the burden of using
animals in pharmacological testing (Selden and
Fuller, 2018). Contemporary bioreactors typically
comprise tri-dimensional cell constructs composed of a solitary phenotype,
co-cultures of diverse phenotypes such as epithelial and endothelial cells, or
epithelial and fibroblastic cells. Alternatively, multiple cell types are
combined to simulate the in vivo microenvironment. Mass transfer enhancement
can be achieved by introducing dynamic characteristics to bioreactors,
specifically through the utilization of convection. This fluid flow mechanism significantly
facilitates mass transfer (Charmet et al., 2020). Various types of bioreactors, such as
spinner flasks, rocking bioreactors, and waveform bioreactors, have the
capacity to achieve a dynamic state through mixing. Nevertheless, these do not
constitute imitations of any physiological system within the human body (Egger
et al., 2017; Birla, 2014). In contrast, perfusion bioreactors provide a more accurate simulation of
the in vivo environment. Micro bioreactors that have achieved success utilize
perfusion systems, which may involve either a basic downward or crossflow
approach or the provision of a microgravity setting. The former mass transfer
method is surpassed by the latter, which utilizes rotating wall cell culture
systems and fluidized bed bioreactors. However, optimizing the flow for
improved tissue-specific expression through optimal perfusion is imperative.
Excessive perfusion can negatively impact cell proliferation, survival, and
function by potentially removing crucial paracrine factors necessary for cell
survival (Irsyad et al., 2022; Nadhif et al., 2020).
Tissue engineering
techniques, such as liver regeneration using cell-based artificial liver
prototypes, are still being developed to address the high demand for liver
transplantation. The need for artificial liver prototypes that are
microstructurally and functionally similar to the liver in vivo presents
challenges in tissue engineering and method refinement. The extracellular
matrix (ECM) microstructure of artificial liver prototypes made from the
natural or synthetic matrix is insufficient for cell proliferation and
differentiation. The decellularized liver scaffold retains the original organ's
ECM, resulting in a scaffold with an ideal microstructure for liver cell
proliferation and differentiation (Antarianto et al., 2022;
Dewi, Antarianto, and Pawitan, 2021). Therefore, artificial liver prototype research is still a
developing area of research, one of which is Stem Cell Hepatic Intuitive
Apparatus (SHiNTA). This study is developed from previous artificial liver
prototype studies (Sibuea et al., 2020). The novelty of this study is the
use of hepatocytes that are differentiated from human induced pluripotent stem
cells (hiPSCs) by using a native liver scaffold with a decellularization
method. The hepatocytes that differentiate from hiPSCs are able to mimic the pattern and
development of hepatocytes through
stages of definitive endoderm differentiation, hepatic specification and
hepatic organ-like maturation (Takeishi et
al., 2020). These methods are made by the Stem
Cell and Tissue Engineering (SCTE) laboratory in IMERI FKUI with multiple
syringe injection methods for the more straightforward manufacturing process as
a central component of SHiNTA (Antarianto et al., 2022;
2019). Another component of
SHiNTA is using a bioreactor made from silicon and a perfusion pump machine
that flows the medium at a specific rate to achieve a higher hepatocyte
maturation and function. The bioreactor's overall design combines with specific
resulted surface roughness produce a distinct perfusion flow can be used to
modulate the perfusion (Qosim et al., 2018; Whulanza
et al., 2016). These
BAL can be used to overcome the problems and weaknesses of existing studies in
the form of price and availability constraints. This research aimed to develop the
SHiNTA BAL for sustaining the viability of hiPSCs-derived hepatocytes in the
artificial liver prototype.
2.1. hiPSCs Culture
In vitronectin-coated (A14700, GibcoTM, USA)
12-well plates, the hiPSCs cell line from human bone marrow mesenchymal stem
cells was purchased from EBiSC (A14700, GibcoTM, USA) was thawed and
cultured with the complete Essential 8 medium culture (A14700, GibcoTM,
USA). Half of the medium was removed every two days and replaced with an equal
volume of fresh medium. Colony morphology and confluency were evaluated daily
through microscopic observation and documentation. The passage was completed
when confluency in each well reached 25–50%. The iPSC colony was washed by
using phosphate-buffered saline (PBS) (10010023, GibcoTM, USA), then
broken up and separated from the vitronectin in the 12-well plates using
Versene solution (15040066, GibcoTM, USA) and incubated for 4-5
minutes at 37°C and 5% CO2. A cell scraper was used to scrap the
hiPSCs from the vitronectin-coated 12-well plates completely. To halt the
dissociative effect, the iPSC suspension was collected in a 5 mL complete
medium with 10 mM ROCK inhibitor (Y-27632, STEMCELL Technologies, Canada). The
split ratio ranged from 1:2 to 1:4.
2.2. Liver scaffold
production and hepatic differentiation to generate artificial liver prototype
Based on
previous research, the researchers utilized decellularization methods to create
a native liver scaffold from the liver of New Zealand White Rabbits through
multiple syringe injections (Antarianto et
al., 2019). Five liver lobules were cut
to 1.5 cm x 1.5 cm with a thickness of 0.7–1 cm. The liver cubes were placed in
a petri dish and immersed in 0.001 M Ethylene Glycol Tetraacetic Acid (EGTA)
(E3889-100G, Merck, Germany) for 30 minutes. A 1 mL syringe was secured with a
fixation device and a toothpick attached to a red wire on top of the Styrofoam.
Aquadest (B000002784, OneMed, Indonesia) was the first administered to liver
cubes using the fixated syringe technique. The sodium dodecyl sulphate (SDS)
(BIO-2050-100g, 1st BASE, Singapore) with graded concentrations of 0.1%, 0.25%,
0.5%, 0.75%, and 1% was administered 25 times at the same location until
translucent. Distilled water was injected into the liver biological scaffolds
to wash, then placed into tubes filled with NaCl 0.9% (GKL9230500149A1, Widatra
Bhakti, Indonesia) solution and stored in the freezer at -80°C.
The biological scaffolds of the liver
were taken out from the freezer and thawed in a biosafety cabinet (BSC) prior
to usage. Using sterile surgical scissors, the scaffold was then cut into three
pieces and placed on a 12-well plate. Before recellularization, the BSC was
sterilized with ultraviolet (UV) light for 1 hour.
The recellularization stage was
completed by injecting 1x106 - 2x106 cells/mL from total
harvested hiPSCs into five pieces of the liver biological scaffolds with a 1 mL
syringe. Based on a modified protocol, the liver scaffold was cultured for 21
days in a static 12-well plate culture with medium changes (Blackford
et al., 2018).
Hepatocyte differentiation induction medium included: complete Essential 8
medium (A1517001, GibcoTM, USA) for days 1-2, RPMI-1640 (RPMI-XRXA,
Capricorn Scientific, Germany) medium with human serum albumin (HSA) 10%
(13533-692-71, Grifols, USA), Antibiotic & Antimycotic 1% (15240062, GibcoTM,
USA), Glutamax 1% (35050061, GibcoTM, USA) for day 4-8, and
HepatoZYME-SFM (17705021, GibcoTM, USA) medium, fetal bovine serum
(FBS) 10% (SH30071.02, Cytiva, USA), Antibiotic & Antimycotic 1% (15240062,
GibcoTM, USA), Glutamax 1% (35050061, GibcoTM, USA) for
day 9-21. Small molecules and growth factors for hepatocyte differentiation
included 1.5 CHIR99021 (72054, STEMCELL Technologies, USA) (day 1), 5 ng/mL
bone morphogenetic protein 4 (BMP4) (PHC9534, GibcoTM, USA) (day
1-2), 5 LY29004 (PHZ1144, GibcoTM, USA) (day 1), 40 ng/mL
fibroblast growth factor 2 (FGF2) (78046, STEMCELL Technologies, USA) (day
1-3), 50 ng/ mL and 25 ng/ mL Activin A (PHC9564, GibcoTM, USA) (day
1-4 for 50 ng/ mL and day 5-8 for 25 ng/ mL), 5 ng/ mL Oncostatin M (OSM)
(PHC5015, GibcoTM, USA) (day 9-21), and 25 ng/ mL hepatocyte growth
factor (HGF) (78019.1, STEMCELL Technologies, USA) (day 9-21). An inverted microscope
was used to observe the scaffold microscopically during the differentiation
process. On day 21, after the differentiation process was completed, the liver
tissue engineering construct samples were collected to initiate the perfusion
process.
2.3. SHiNTA artificial liver
system prototyping
This research was consisted of four
groups: Group 1 was SHiNTA BALs perfusion with the
bioreactor for seven days (SH-H7), Group 2 was SHiNTA BALs inside the blood bag on top of
orbital shaker for seven days
(BL-H7), Group 3 was decellularized native liver scaffold only as a negative
control (K-), and Group 4 was artificial liver prototype as a positive control
(K+).
The
SHiNTA BALs perfusion with the bioreactor group (SH-H7 group) was placed in a
bioreactor made of polydimethylsiloxane according to previous studies (Irsyad
et al., 2022; Sagita et al., 2018; Whulanza et al., 2016). A closed perfusion circuit was
created with the installation of 3 mm silicon tubes in the bioreactor, a blood
bag contained with complete hepatocyte medium, and a perfusion pump (Perista
Pump SJ-1211, Atto, Japan), as shown in the illustration in Figure 1. Complete
hepatocyte medium consists of Williams's E medium (12551032, GibcoTM,
USA) with platelet-rich plasma (PRP) 10% provided from Palang Merah Indonesia
(PMI), dexamethasone 1% (GKL8619906143A1, Phapros, Indonesia),
Insulin-Transferrin-Selenium (ITS) 1% (41400045, GibcoTM, USA),
Antibiotic & Antimycotic 1% (15240062, GibcoTM, USA), Glutamax
1% (35050061, GibcoTM, USA), and heparin 1% (GKL1731539143A1,
Fahrenheit, USA) (Sibuea et al., 2020). Perfusion pumps were run for seven
days with a perfusion rate of 2 mL/min. Samples were collected on day 7 after
the perfusion process was completed for histological analyses.
Figure 1 (a) An illustration of a closed-circuit SHiNTA with a
bioreactor inside an incubator. Arrow: Flow direction of complete hepatocyte
medium (b) An illustration of cell distribution inside a liver scaffold
2.4. Cell counting and viability
analysis
The
preparations of cell viability in liver tissue engineering construct groups were made
with a working solution of Live/Dead Viability/Cytotoxicity Kit (L3224,
Invitrogen, USA) by combining Ethidium Homodimer-1 with Dulbecco's phosphate-buffered
saline (DPBS) (14190144, GibcoTM, USA) and Calcein AM (4:2). Samples were fixated with methanol
for 30 minutes, then added with 100-150 µl working solution, and incubated in a
moist chamber at dark room for 30 minutes. After incubation,
samples were added with DPBS. The viability and cell counting results were
examined using Biotek Cytation1 Image Reader (Agilent, USA) at Integrity
Laboratory and Research Center (ILRC), Universitas Indonesia.
2.5. Histological analysis for cell adhesion to extracellular matrix in scaffold and measurement of
collagen area fraction
Haematoxylin and eosin (HE) staining
was carried out by deparaffinization with xylol (1.08661.2500, Merck, Germany)
and rehydration in decreasing graded alcohol concentrations (1.00983.2500,
Merck, Germany) (100%, 96%, 80%, and 70 %). After being treated in a
hematoxylin solution (3801562, Leica, USA) for 10-15 minutes, the samples were
washed under running water and then incubated in eosin (3801602, Leica, USA)
for 5-10 seconds. The samples were dehydrated with increasing alcohol
concentrations. (70%, 80%, 96%, and 100%) and cleared with xylol. After
clearing, the samples were covered with a cover slip and enclosed with Entelan
(1.07960, Merck, Germany).
Masson’s Trichrome (MT) staining was
carried out by deparaffinized with xylol and rehydration in decreasing graded
alcohol concentrations (100%, 96%, 80%, and 70%). The samples were fixated in
Bouin's fixative solution (HT10132, Merck, Germany) for one hour at 56°C, then
washed in running water, submerged in Weigert hematoxylin solution (115973,
Merck, Germany) (30 minutes), Biebrich scarlet acid fuchsin (HT151, Merck,
Germany) (five minutes), phosphomolybdic acid (HT153, Merck, Germany) (five
minutes), and aniline blue (B8563, Merck, Germany) (30 minutes). Tissue samples were dehydrated with
increasing alcohol concentrations (70%, 80%, 96%, and 100%), followed by xylol
clearance. . After clearing, the samples were covered with a cover slip and
enclosed with Entelan. To calculate the fraction of collagen area relative to
scaffold area on MT staining, ImageJ software was used to evaluate the micro
photo findings from the Optilab, with replication of scaffold area photo taken
three times per group sample (n=3).
2.6. Immunofluorescence
analysis of
artificial liver prototype
The immunofluorescence technique was
conducted to investigate the expression of hepatocyte transcription factors in
the liver tissue engineering construct groups. The samples were deparaffinized
using xylol and rehydrated through a series of decreasing graded alcohol
concentrations (100%, 96%, 80%, and 70%). The samples were then incubated with
primary antibodies, including anti-HNF4- (ab92378 Abcam, UK) at a 1:100
dilution, anti-CEBP- (ab40761 Abcam, UK) at a 1:250 dilution, and BD PharmigenTM
PE Mouse anti-ASGPR1 (Clone 8D7, BD Biosciences, USA) at a 1:100 dilution. The
incubation procedure for anti-HNF4- and anti-CEBP- was carried out in a moist
chamber at a temperature of 4°C for the night. The incubation process for
anti-ASGPR1 was carried out in a moist chamber and dark room at room
temperature for two hours in the absence of secondary antibody incubation. The
experimental procedure involved a washing step with 0.1% PBST, followed by
incubation of Goat anti-Rabbit IgG (H+L) Cross-Adsorbed secondary antibody
Alexa FluorTM 488 (A-11008, Invitrogen, USA) in 1:200 dilution for
anti-HNF4- and anti-CEBP-. The secondary antibody was incubated for one hour
at room temperature in a dark room. Following the second antibody incubation,
the samples were washed with 0.1% PBST. Subsequently, the samples were
incubated with 4’,6-diamidino-2-phenylindole (DAPI) (ab228549, Abcam, UK)
staining for five minutes at room temperature in a dark room. Following the
immunofluorescence staining procedure, the samples were subjected to analysis
utilizing a ZOETM Fluorescent Cell Imager (Bio-Rad, USA) at the
Gerontology laboratory IMERI FKUI.
2.7. Data analysis
Data analysis in this study was carried
out by statistical tests using GraphPad PRISM Version 9 software. A statistical
analysis was performed using quantitative histological analysis using ImageJ
software (collagen area). The normality test was carried out using the
Shapiro-Wilk test, and the homogeneity test was carried out using the Levene
test. The P value <.05 indicates a significant difference between the two
groups.
Results
3.1. SHiNTA
bioreactor perfusion
The configuration of the SHiNTA BALs with perfusion
(SH-H7) group study comprises a blood bag containing complete hepatocyte medium
attached to a perfusion pump, demonstrating the capability for perfusion. The
perfusion did not cause the sample in the bioreactor chamber to rupture or be
forced into tiny tubes during the seven days of perfusion with electrical power
application. The 2 mL/min perfusion rate allows for
appropriate perfusion while subjecting the sample to the least degree of shear
stress throughout its stay in the bioreactor chamber. In contrast, the
orbital shaker methods used in the SHiNTA blood bag (BL-H7) group trials showed
that the samples did not rupture throughout the seven days. Each group's artificial liver prototype can be
used for histopathological and molecular examination.
3.2. SHiNTA
histological staining
Figure 2 Histological features of SHiNTA using HE staining
(Mag. 40x): (a) SHiNTA BALs perfusion with
the bioreactor for seven days
(SH-H7), (b) SHiNTA BALs inside the blood bag on top of orbital shaker for
seven days (BL-H7), (c) artificial liver prototype as a positive control (K+),
and (d) decellularized native liver scaffold only as a negative control (K-)
According to findings from
Figure 3a's Masson’s Trichrome (MT) staining, cells in the SH-H7 group had
eosinophilic cytoplasm and blue-black nuclei. They could be visible in the
pores of the scaffold or on the collagen fibers' surface. As illustrated in Figure
3b, the cells in the BL-H7 group could also be observed on the surface of the
collagen fibers or by filling the holes of the scaffold. The ECM in the
negative control (K-) group of decellularized liver scaffolds (Figure 3d) was
made up of blue-colored collagen fibers with voids between them (which appeared
as hollow white spaces between fibers). In K+ group of liver tissue engineering
construct in 21 days showed the cells on the surface of collagen fibers of ECM
(Figure 3c).