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Development of liver decellularized extracellular matrix bioink for 3D cell printing-based liver tissue engineering Hyungseok Lee, Wonil Han, Hyeonji Kim, Dong-Heon Ha, Jinah Jang, Byoung Soo Kim, and Dong-Woo Cho Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01908 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017
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Biomacromolecules
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Development of liver decellularized extracellular matrix
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bioink for 3D cell printing-based liver tissue engineering
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Hyungseok Lee1,*, Wonil Han2,*, Hyeonji Kim1,
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Dong-Heon Ha1, Jinah Jang3, Byoung Soo Kim1, and Dong-Woo Cho1, †
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San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, South Korea
Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH),
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y (POSTECH), 77 Cheongam ro, Nam-gu, Pohang, Kyungbuk 790-784, South Korea.
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San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, South Korea
Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technolog
Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH),
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* Hyungseok Lee and Wonil Han contributed equally to this article.
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†
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Dong-Woo Cho, Ph.D.
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Department of Mechanical Engineering, POSTECH
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San 31 Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, South Korea
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Tel.: +82 54 279 2171; Fax: +82 54 279 5419; E-mail address:
[email protected] Corresponding author
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Abstract
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The liver is an important organ and plays major roles in the human body. Due to the lack of
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liver donors after liver failure and drug-induced liver injury, much research has focused on
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developing liver alternatives and liver in vitro models for transplantation and drug screening.
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Although numerous studies have been conducted, these systems cannot faithfully mimic the
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complexity of the liver. Recently, three-dimensional (3D) cell printing technology has
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emerged as one of a number of innovative technologies that may help to overcome this
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limitation. However, great scope remains in developing biomaterials optimized for 3D cell
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printing-based liver tissue engineering. Therefore, in this research, we developed a liver
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decellularized extracellular matrix (dECM) bioink for 3D cell printing applications and
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evaluated its characteristics. The liver dECM bioink retained the major ECM components of
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the liver while cellular components were effectively removed, and further exhibited suitable
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and adjustable properties for 3D cell printing. We further studied printing parameters with the
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liver dECM bioink to verify the versatility and fidelity of the printing process. Stem cell
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differentiation and HepG2 cell functions in the liver dECM bioink in comparison to
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commercial collagen bioink were also evaluated, and the liver dECM bioink was found to
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induce stem cell differentiation and enhance HepG2 cell function. Consequently, the results
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demonstrate that the proposed liver dECM bioink is a promising bioink candidate for 3D cell
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printing-based liver tissue engineering.
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Keywords: 3D cell printing technology, liver, tissue engineering, decellularized extracellular
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matrix (dECM), bioink
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1. Introduction
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The liver is a vital organ which plays major roles in balancing biochemical environments in
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the human body, including blood protein synthesis, glucose metabolism, and detoxification of
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metabolites or other chemicals.1 Liver transplantation is widely used in the case of liver
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failure; however, transplantation is limited by a lack of liver donors.2, 3 Furthermore, many
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pharmaceutical products have been banned or not approved because of drug-induced liver
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injury.4 Numerous studies have focused on developing liver alternatives and liver in vitro
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models. Takabe et al. have developed a vascularized functional human liver using induced
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pluripotent stem cells,5 and a recellularized liver graft was developed from a decellularized
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liver matrix by Uygun et al.6 Additionally, liver spheroids have been studied as a liver in vitro
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model for drug screening.7-9 Although various studies have been conducted to develop liver
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alternatives and liver in vitro models, the aforementioned studies could not control the
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positions of the multiple cell types and of the extracellular matrix (ECM) of the liver.
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Furthermore, it is nearly impossible to prepare patient-specific liver constructs due to a lack
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of fabrication methods.
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Three-dimensional (3D) cell printing is an emerging technology in bioengineering due to its
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suitability for the fabrication of complex 3D biological constructs.10, 11 The most promising
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advance in 3D cell printing is that various biomaterials and multiple cell types can be printed
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simultaneously as intended to fabricate complex biological structures.12, 13 Taking advantage
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of these advances, Kang et al. have applied 3D cell printing to produce human-scale tissues,
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and they have also shown that it is possible to achieve patient specificity.14 Further to
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advances in 3D cell printing, the development of bioink, which encapsulates the cells during
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the printing process, is another key means of improving the functionality of cell printed 3
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constructs. Several studies have focused on liver bioink development. Mixtures, such as
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gelatin-chitosan, agarose-collagen, and agarose-chitosan, have been employed, for example.15,
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16
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remains to be explored. As per another example, when the liver-derived material was used as
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a liver bioink, the printing uniformity of the bioink had to be improved using poly(ethylene
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glycol) diacrylate (PEGDA) and an ultraviolet (UV) crosslinking process.17 Occasionally, UV
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light or radicals associated with the photoinitiator affect cell behavior unexpectedly. As these
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examples show, much effort has gone into the development of the liver bioink; however, no
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study has successfully produced a liver-derived bioink that is non-toxic and offers adjustable
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stiffness and high printability.
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In this study, we developed and investigated the characteristics and performance of a liver
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decellularized extracellular matrix (dECM) bioink for 3D cell printing. Initially, we evaluated
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the biochemical characteristics of the liver dECM bioink after the decellularization process.
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Then, its rheological properties, printing parameters, and cytotoxicity as well as printed
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constructs were assessed. Furthermore, stem cell differentiation and the functionality of
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HepG2 cells in the liver dECM bioink were evaluated and compared with those in
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commercial collagen bioink. The liver dECM bioink developed here improved the
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functionality of printed structures.
However, these bioinks did not contain liver-specific biochemical components, which
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2. Materials and Methods
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2.1. Liver decellularization
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Porcine liver tissue was sliced roughly into 0.5–1-mm-thick slices and washed with distilled 4
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water for 2 hours. Next, the slices were treated with 0.5% Triton X-100 (Sigma-Aldrich, USA)
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in 1 M NaCl (Samchun Pure Chemicals, Korea) for 9 hours. Any remaining cellular
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components were washed out with 1% SDS (Affymetrix, USA) for 3 hours, and the sample
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was sterilized by means of a 1-hour treatment with 0.1% peracetic acid (Sigma-Aldrich, USA)
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in phosphate-buffered saline (PBS). Between decellularization steps, the tissues were washed
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with distilled water for 2-5 hours to remove remaining detergent and cell debris.
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Decellularized tissues were freeze-dried for 48 hours with Free Zone 2.5 (Labconco, USA)
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and then used for biochemical characterization and liver dECM bioink preparation.
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2.2. Biochemical characterization of liver dECM
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To quantify double-stranded DNA content, a DNA quantification kit (Sigma-Aldrich, USA)
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was purchased and assays were conducted. Deoxyribonucleic acid sodium salt (Sigma-
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Aldrich, USA) was used for the standard sample of double-stranded DNA. Fluorescence
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intensity was measured with a SpectraMax Gemini XPS (Molecular Devices, excitation
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wavelength: 360nm, emission wavelength: 450nm). For the immunohistochemical analysis,
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native liver and decellularized tissues were fixed in 4% formalin, embedded in paraffin, and
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sectioned with a microtome. Sectioned samples were stained with hematoxylin and eosin
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(H&E), Masson's trichrome, anti-fibronectin, and alcian blue, and then the stained samples
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were examined with a light microscope.
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2.3. Preparation of biomaterials
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To prepare the liver dECM bioink for the 3D cell printing system, the liver dECM was
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lyophilized for 24 hours and ground into powder with a grinder. The liver dECM powder was
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dissolved in 0.5M acetic acid (Duksan, Korea) with 10% tissue weight of pepsin (Sigma-
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Aldrich, USA) using a magnetic stirrer for 4 days. The liver dECM solution was centrifuged 5
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at 3500 rpm for 10 min to remove large particles. Finally, to prepare the liver dECM bioink,
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the pH of the liver dECM solution was adjusted to 7.4 with 10 M sodium hydroxide. Porcine
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skin-derived collagen type I (Dalim Tissen, Korea) was dissolved in 0.5 M acetic acid for
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four days and neutralized with 10 M sodium hydroxide for the collagen bioink. The liver
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dECM and collagen bioink were prepared and maintained at below 4 °C. Finally,
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polycaprolactone (PCL) was also purchased from Polysciences (USA).
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2.4. Rheological characterization
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The rheological characteristics of the 1.5% (15 mg/ml) and 3% (30 mg/ml) liver dECM
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bioinks and collagen bioinks in the same concentrations were evaluated with the Advanced
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Rheometric Expansion System (TA Instruments, USA). A steady shear sweep analysis of the
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liver dECM collagen bioinks was performed at 15 °C to evaluate its viscosity. A dynamic
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frequency sweep analysis was conducted to measure the frequency-dependent storage (G’)
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and loss (G”) modulus of the liver dECM and collagen bioinks.
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2.5. Cell preparation and encapsulation in bioinks
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Human hepatocellular carcinoma (HepG2) cell lines and human bone marrow-derived
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mesenchymal stem cells (BMMSCs) were purchased from ATCC (USA) and Cefobio (Seoul,
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Korea) respectively. Both cell types were cultured in Dulbecco’s modified Eagle’s medium
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(DMEM, Gibco BRL, NY, USA) with 10% (v/v) fetal bovine serum (FBS, Gibco BRL, NY,
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USA) and 1% (v/v) penicillin/streptomycin (P/S, Sigma-Aldrich, St. Louis, MO, USA) at
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37 °C in a humidified 5% CO2 atmosphere. All cells used were from passage 2 to 4.
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BMMSCs and HepG2 cells were encapsulated in bioink separately, at a concentration of 5 x
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106 cells/ml.
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2.6. Application to 3D cell printing technology
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To study the 3D cell printing process with the liver dECM bioink, the printing parameters of
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pneumatic pressure, printing speed, and nozzle diameter were altered. PCL material was
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printed with liver dECM bioink to make the hybrid constructs, and PCL was printed at a
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temperature of 60 °C and a pneumatic pressure of 500 kPa. After the printing process, the
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printed structures were incubated for 1 hour for gelation. All printing procedures were carried
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out with an in-house-developed 3D cell-printing system designed to print polymers and
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bioinks using pneumatic pressure and heat induced by a heating controller.18 The heating
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temperature and pneumatic pressure can respectively be raised to 150 °C and 650 kPa.
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2.7. Culture of cell printed constructs
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The BMMSC and HepG2 cell-printed constructs of 2D patterns were cultured in Dulbecco’s
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modified Eagle’s medium (DMEM, Gibco BRL, NY, USA) with 10% (v/v) fetal bovine
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serum (FBS, Gibco BRL, NY, USA) and 1% (v/v) penicillin/streptomycin (P/S, Sigma-
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Aldrich, St. Louis, MO, USA) at 37 °C in a humidified 5% CO2 atmosphere. No supplements
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were treated to analyze the biochemical functionalities of liver dECM bioink alone compared
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to the collagen bioink, which was used as a negative control. The culture media was
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exchanged every two to three days, and samples were properly harvested in accordance with
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the analysis.
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2.8. Evaluation of cell viability
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To assess the viability of the HepG2 and BMMSCs cells after the 3D cell printing process,
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samples were stained with live/dead kits (Life Technologies, Germany). 4mM calcein AM for
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the live cell assay and 2mM ethidium homodimer solution for the dead cell assay were mixed 7
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in a ratio of 1 to 4 in PBS and incubated with printed samples at 37 °C for 30 min. This was
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followed by PBS washing to remove any remainders of the solutions. Live and dead cells
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were visualized with a Zeiss LSM 510 Meta confocal microscope (Zeiss, Jena, Germany).
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2.9. Gene expression analysis
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Samples of printed structure were harvested, and total RNA from each sample was isolated
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using the RNeasy Isolation Kit (QIAGEN) according to the manufacturer’s instructions.
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Complementary DNA was synthesized using a Maxima First Strand cDNA Synthesis Kit
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(Thermo Scientific, Waltham, MA, USA). Gene expression was analyzed with SYBR green
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PCR Master Mix using real-time PCR (Applied Biosystems, Foster City, CA, USA).
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The mRNA sequence of target genes was obtained from the National Center for
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Biotechnology Information (NCBI) gene sequence database, and all primers (Table 1) were
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designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA).
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Forward and reverse primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH),
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albumin (ALB), alpha-fetoprotein (AFP), transthyretin (TTR), cytokeratin 18 (CK18),
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hepatocyte nuclear factor 1 alpha (HNF1A), hepatocyte nuclear factor 3 beta (HNF3B),
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hepatocyte nuclear factor 4 alpha (HNF4A), and hepatocyte nuclear factor 6 (HNF6) were
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purchased from Pioneer (Korea). Gene expression levels were all normalized relative to
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GAPDH.
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Table 1. Primer sequences for quantitative real-time PCR analysis. Gene GAPDH HNF1α HNF3β
Sense Anti-sense Sense Anti-sense Sense Anti-sense
Sequence ATGGAAATCCCATCACCATCTT CGCCCCACTTGATTTTGG GAGGAGCGAGAGACGCTAGTG CACCCCTCTCTGGATGCATT CTGAAGCCGGAACACCACTAC CGAGGACATGAGGTTGTTGATG 8
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HNF4α HNF6 ALB AFP CK18 TTR
Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense Sense Anti-sense
CGCTACTGCAGGCTCAAGAAA TCTGGACGGCTTCCTTCTTC TGCGCAACCCCAAACC TCCACATCCTCCGGAAGGT TTGCATGAGAAAACGCCAGTA AGCATGGTCGCCTGTTCAC TCGGACACTTATGTATCAGACATGAA GCCTCCTGTTGGCATATGAAG GCCTTGGACAGCAGCAACTC ACCACTTTGCCATCCACTATCC ACCAAATCTTACTGGAAGGCACTT GAGTCGTTGGCTGTGAATACCA
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2.10. Immunofluorescence staining
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The 3D cell printed liver constructs were fixed with 4% paraformaldehyde. The samples were
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permeabilized with 0.1% Triton X-100 and were treated with 3% bovine serum albumin
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(Affimetrix, USA) in PBS for 1 hour to block the nonspecific binding. The samples were
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washed with PBS three times for 15 min. Anti-human HNF4A rabbit antibody (Abcam, UK)
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and anti-human AFP mouse antibody (Abcam, UK) were used as primary antibodies and
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treated overnight at 4 °C. After washing with PBS, the samples were treated with Alexa Fluor
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594 goat anti-rabbit antibody (Invitrogen, CA, USA) and Alexa Fluor 488 goat anti-mouse
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antibody (Invitrogen, CA, USA) for 1 hour at 37 °C and counterstained with 4',6-diamidino-
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2-phenylindole (DAPI). Stained images were obtained with a FV1000 Olympus confocal
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microscope (Olympus, Tokyo).
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2.11. Liver function test
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Constructs fabricated with collagen and the liver dECM bioink with the same concentration
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of 1.5% were cultured for seven days to conduct a liver function test. Medium samples were
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collected for analysis for secreted albumin/urea and refreshed every three days. Secreted
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albumin was quantified with an enzyme-linked immunosorbent assay (ELISA) (DuoSet 9
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ELISA development system, Genzyme). Secreted urea was quantified with a urea assay kit
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(BioVision, USA). Quantification was performed with a microplate reader (Epoch 2
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Microplate Spectrophotometer, BioTek, USA).
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2.12. Statistical analysis
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All variables for liver differentiation and liver-specific functions were expressed as means ±
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standard deviations (SDs). Evaluation of the difference between the mean values of each
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group was performed by Student’s t-test, in which a p-value
