Article pubs.acs.org/Biomac
Development of Liver Decellularized Extracellular Matrix Bioink for Three-Dimensional Cell Printing-Based Liver Tissue Engineering Hyungseok Lee,† Wonil Han,‡ Hyeonji Kim,† Dong-Heon Ha,† Jinah Jang,§ Byoung Soo Kim,† and Dong-Woo Cho*,†
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Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, South Korea ‡ Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), 77 Cheongam ro, Nam-gu, Pohang, Kyungbuk 790-784, South Korea § Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, South Korea ABSTRACT: The liver is an important organ and plays major roles in the human body. Because of the lack of liver donors after liver failure and drug-induced liver injury, much research has focused on developing liver alternatives and liver in vitro models for transplantation and drug screening. Although numerous studies have been conducted, these systems cannot faithfully mimic the complexity of the liver. Recently, three-dimensional (3D) cell printing technology has emerged as one of a number of innovative technologies that may help to overcome this limitation. However, a great deal of work in developing biomaterials optimized for 3D cell printing-based liver tissue engineering remains. Therefore, in this work, we developed a liver decellularized extracellular matrix (dECM) bioink for 3D cell printing applications and evaluated its characteristics. The liver dECM bioink retained the major ECM components of the liver while cellular components were effectively removed and further exhibited suitable and adjustable properties for 3D cell printing. We further studied printing parameters with the liver dECM bioink to verify the versatility and fidelity of the printing process. Stem cell differentiation and HepG2 cell functions in the liver dECM bioink in comparison to those of commercial collagen bioink were also evaluated, and the liver dECM bioink was found to induce stem cell differentiation and enhance HepG2 cell function. Consequently, the results demonstrate that the proposed liver dECM bioink is a promising bioink candidate for 3D cell printingbased liver tissue engineering.
1. INTRODUCTION
it is nearly impossible to prepare patient-specific liver constructs because of a lack of fabrication methods. Three-dimensional (3D) cell printing is an emerging technology in bioengineering because of its suitability for the fabrication of complex 3D biological constructs.10,11 The most promising advance in 3D cell printing is that various biomaterials and multiple cell types can be printed simultaneously as intended to fabricate complex biological structures.12,13 Taking advantage of these advances, Kang et al. have applied 3D cell printing to produce human-scale tissues, and they have also shown that it is possible to achieve patient specificity.14 With respect to advances in 3D cell printing, the development of bioink, which encapsulates the cells during the printing process, is another key means of improving the functionality of cell-printed constructs. Several studies have focused on liver bioink development. Mixtures, such as gelatin
The liver is a vital organ that plays major roles in balancing biochemical environments in the human body, including blood protein synthesis, glucose metabolism, and detoxification of metabolites or other chemicals.1 Liver transplantation is widely used in the case of liver failure; however, transplantation is limited by a lack of liver donors.2,3 Furthermore, many pharmaceutical products have been banned or not approved because of drug-induced liver injury.4 Numerous studies have focused on developing liver alternatives and liver in vitro models. Takabe et al. have developed a vascularized functional human liver using induced pluripotent stem cells,5 and a recellularized liver graft was developed from a decellularized liver matrix by Uygun et al.6 Additionally, liver spheroids have been studied as a liver in vitro model for drug screening.7−9 Although various studies have been conducted to develop liver alternatives and liver in vitro models, the aforementioned studies could not control the positions of the multiple cell types and of the extracellular matrix (ECM) of the liver. Furthermore, © 2017 American Chemical Society
Received: December 23, 2016 Revised: March 8, 2017 Published: March 9, 2017 1229
DOI: 10.1021/acs.biomac.6b01908 Biomacromolecules 2017, 18, 1229−1237
Article
Biomacromolecules
Instruments). A steady shear sweep analysis of the liver dECM collagen bioinks was performed at 15 °C to evaluate its viscosity. A dynamic frequency sweep analysis was conducted to measure the frequency-dependent storage (G′) and loss (G″) modulus of the liver dECM and collagen bioinks. 2.5. Cell Preparation and Encapsulation in Bioinks. Human hepatocellular carcinoma (HepG2) cell lines and human bone marrow-derived mesenchymal stem cells (BMMSCs) were purchased from ATCC and Cefobio (Seoul, Korea), respectively. Both cell types were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) with 10% (v/v) fetal bovine serum (FBS, Gibco BRL) and 1% (v/v) penicillin/streptomycin (P/S, Sigma-Aldrich, St. Louis, MO) at 37 °C in a humidified 5% CO2 atmosphere. All cells used were from passage 2 to 4. BMMSCs and HepG2 cells were encapsulated in bioink separately, at a concentration of 5 × 106 cells/mL. 2.6. Application to 3D Cell Printing Technology. To study the 3D cell printing process with the liver dECM bioink, printing parameters such as pneumatic pressure, printing speed, and nozzle diameter were altered. PCL material was printed with liver dECM bioink to make the hybrid constructs, and PCL was printed at a temperature of 60 °C and a pneumatic pressure of 500 kPa. After the printing process, the printed structures were incubated for 1 h for gelation. All printing procedures were performed with an in-housedeveloped 3D cell printing system designed to print polymers and bioinks using pneumatic pressure and heat induced by a heating controller.18 The heating temperature and pneumatic pressure can be increased to 150 °C and 650 kPa, respectively. 2.7. Culture of Cell-Printed Constructs. The BMMSC and HepG2 cell-printed constructs of two-dimensional (2D) patterns were cultured in DMEM (Gibco BRL) with 10% (v/v) FBS (Gibco BRL) and 1% (v/v) P/S (Sigma-Aldrich) at 37 °C in a humidified 5% CO2 atmosphere. No supplements were added to analyze the biochemical functionalities of liver dECM bioink alone compared to the collagen bioink, which was used as a negative control. The culture medium was exchanged every 2−3 days, and samples were properly harvested in accordance with the analysis. 2.8. Evaluation of Cell Viability. To assess the viability of the HepG2 and BMMSCs cells after the 3D cell printing process, samples were stained with live/dead kits (Life Technologies); 4 mM calcein AM for the live cell assay and a 2 mM ethidium homodimer solution for the dead cell assay were mixed in a 1:4 ratio in PBS and incubated with printed samples at 37 °C for 30 min. This was followed by PBS washing to remove any remaining solution. Live and dead cells were visualized with a Zeiss LSM 510 Meta confocal microscope (Zeiss, Jena, Germany). 2.9. Gene Expression Analysis. Samples of printed structure were harvested, and total RNA from each sample was isolated using the RNeasy Isolation Kit (QIAGEN) according to the manufacturer’s instructions. Complementary DNA was synthesized using a Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). Gene expression was analyzed with SYBR green PCR Master Mix using real-time polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA). The mRNA sequence of target genes was obtained from the National Center for Biotechnology Information (NCBI) gene sequence database, and all primers (Table 1) were designed using Primer Express version 3.0 (Applied Biosystems). Forward and reverse primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), albumin (ALB), α-fetoprotein (AFP), transthyretin (TTR), cytokeratin 18 (CK18), hepatocyte nuclear factor 1α (HNF1A), hepatocyte nuclear factor 3β (HNF3B), hepatocyte nuclear factor 4α (HNF4A), and hepatocyte nuclear factor 6 (HNF6) were purchased from Pioneer. Gene expression levels were all normalized relative to GAPDH. 2.10. Immunofluorescence Staining. The 3D cell-printed liver constructs were fixed with 4% paraformaldehyde. The samples were permeabilized with 0.1% Triton X-100 and treated with 3% bovine serum albumin (Affimetrix) in PBS for 1 h to block the nonspecific binding. The samples were washed three times with PBS for 15 min. Anti-human HNF4A rabbit antibody (Abcam) and anti-human AFP
and chitosan, agarose and collagen, and agarose and chitosan, have been employed, for example.15,16 However, these bioinks did not contain liver-specific biochemical components, which remains to be explored. As per another example, when the liverderived material was used as a liver bioink, the printing uniformity of the bioink had to be improved using poly(ethylene glycol) diacrylate (PEGDA) and an ultraviolet (UV) cross-linking process.17 Occasionally, UV light or radicals associated with the photoinitiator affect cell behavior unexpectedly. As these examples show, much effort has gone into the development of the liver bioink; however, no study has successfully produced a liver-derived bioink that is nontoxic and offers adjustable stiffness and high printability. In this study, we developed and investigated the characteristics and performance of a liver decellularized extracellular matrix (dECM) bioink for 3D cell printing. Initially, we evaluated the biochemical characteristics of the liver dECM bioink after the decellularization process. Then, its rheological properties, printing parameters, and cytotoxicity as well as printed constructs were assessed. Furthermore, stem cell differentiation and the functionality of HepG2 cells in the liver dECM bioink were evaluated and compared with those in commercial collagen bioink. The liver dECM bioink developed here improved the functionality of printed structures.
2. MATERIALS AND METHODS 2.1. Liver Decellularization. Porcine liver tissue was sliced roughly into 0.5−1 mm thick slices and washed with distilled water for 2 h. Next, the slices were treated with 0.5% Triton X-100 (SigmaAldrich) in 1 M NaCl (Samchun Pure Chemicals) for 9 h. Any remaining cellular components were washed out with 1% sodium dodecyl sulfate (Affymetrix) for 3 h, and the sample was sterilized by means of a 1 h treatment with 0.1% peracetic acid (Sigma-Aldrich) in phosphate-buffered saline (PBS). Between decellularization steps, the tissues were washed with distilled water for 2−5 h to remove the remaining detergent and cell debris. Decellularized tissues were freezedried for 48 h with Free Zone 2.5 (Labconco) and then used for biochemical characterization and liver dECM bioink preparation. 2.2. Biochemical Characterization of Liver dECM. To quantify the double-stranded DNA content, a DNA quantification kit (SigmaAldrich) was purchased, and assays were conducted. Deoxyribonucleic acid sodium salt (Sigma-Aldrich) was used for the standard sample of double-stranded DNA. The fluorescence intensity was measured with a SpectraMax Gemini XPS instrument (Molecular Devices, excitation wavelength of 360 nm, emission wavelength of 450 nm). For the immunohistochemical analysis, native liver and decellularized tissues were fixed in 4% formalin, embedded in paraffin, and sectioned with a microtome. Sectioned samples were stained with hematoxylin and eosin (H&E), Masson’s trichrome, anti-fibronectin, and alcian blue, and then the stained samples were examined with a light microscope. 2.3. Preparation of Biomaterials. To prepare the liver dECM bioink for the 3D cell printing system, the liver dECM was lyophilized for 24 h and ground into powder with a grinder. The liver dECM powder was dissolved in 0.5 M acetic acid (Duksan) with a 10% tissue weight of pepsin (Sigma-Aldrich) using a magnetic stirrer for 4 days. The liver dECM solution was centrifuged at 3500 rpm for 10 min to remove large particles. Finally, to prepare the liver dECM bioink, the pH of the liver dECM solution was adjusted to 7.4 with 10 M sodium hydroxide. Porcine skin-derived collagen type I (Dalim Tissen) was dissolved in 0.5 M acetic acid for 4 days and neutralized with 10 M sodium hydroxide for the collagen bioink. The liver dECM and collagen bioink were prepared and maintained below 4 °C. Finally, polycaprolactone (PCL) was also purchased from Polysciences. 2.4. Rheological Characterization. The rheological characteristics of the 1.5% (15 mg/mL) and 3% (30 mg/mL) liver dECM bioinks and collagen bioinks at the same concentrations were evaluated with the Advanced Rheometric Expansion System (TA 1230
DOI: 10.1021/acs.biomac.6b01908 Biomacromolecules 2017, 18, 1229−1237
Article
Biomacromolecules
group was performed with a Student’s t test, in which a p value of
