Article pubs.acs.org/Biomac
Liver Extracellular Matrix Providing Dual Functions of TwoDimensional Substrate Coating and Three-Dimensional Injectable Hydrogel Platform for Liver Tissue Engineering Jung Seung Lee,† Jisoo Shin,† Hae-Min Park,‡ Yun-Gon Kim,§ Byung-Gee Kim,‡ Jong-Won Oh,† and Seung-Woo Cho*,† †
Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Republic of Korea § Department of Chemical Engineering, Soongsil University, Seoul 156-743, Republic of Korea
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ABSTRACT: Decellularization of tissues or organs can provide an efficient strategy for preparing functional scaffolds for tissue engineering. Microstructures of native extracellular matrices and their biochemical compositions can be retained in the decellularized matrices, providing tissue-specific microenvironments for efficient tissue regeneration. Here, we report the versatility of liver extracellular matrix (LEM) that can be used for two-dimensional (2D) coating and three-dimensional (3D) hydrogel platforms for culture and transplantation of primary hepatocytes. Collagen type I (Col I) has typically been used for hepatocyte culture and transplantation. In this study, LEM was compared with Col I in terms of biophysical and mechanical characteristics and biological performance for enhancing cell viability, differentiation, and hepatic functions. Surface properties of LEM coating and mechanical properties and gelation kinetics of LEM hydrogel could be manipulated by adjusting the LEM concentration. In addition, LEM hydrogel exhibited improved elastic properties, rapid gelation, and volume maintenance compared to Col I hydrogel. LEM coating significantly improved hepatocyte functions such as albumin secretion and urea synthesis. More interestingly, LEM coating upregulated hepatic gene expression of human adipose-derived stem cells, indicating enhanced hepatic differentiation of these stem cells. The viability and hepatic functions of primary hepatocytes were also significantly improved in LEM hydrogel compared to Col I hydrogel both in vitro and in vivo. Albumin and hepatocyte transcription factor expression was upregulated in hepatocytes transplanted in LEM hydrogels. In conclusion, LEM can provide functional biomaterial platforms for diverse applications in liver tissue engineering by promoting survival and maturation of hepatocytes and hepatic commitment of stem cells. This study demonstrates the feasibility of decellularized matrix for both 2D coating and 3D hydrogel in liver tissue engineering.
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protein in the liver that can interact with hepatocytes,4,5 has been used for surface coating and hydrogel formation to culture and transplant hepatocytes.6−8 Unfortunately, currently available collagen-based methods need to be further improved to maintain the viability and hepatic functions of primary hepatocytes.9 Therefore, functional substrates and scaffolds capable of providing in vivo-like microenvironments favorable for hepatocytes should be developed for application of
INTRODUCTION Orthotopic liver transplantation has been used for treating endstage liver diseases caused by chronic cirrhosis and cancer and restoring functions of severely damaged liver. However, transplantation is limited due to a shortage of liver donors. Transplantation of hepatic lineage cells (e.g., hepatocytes) has been tested as an alternative to liver transplantation.1,2 However, this method also suffers from practical limitations due mainly to the difficulty in obtaining sufficient numbers of functional hepatocytes for therapeutic efficacy. Furthermore, primary hepatocytes easily lose viability and hepatic functions during in vitro culture or after transplantation.3 To overcome these limitations, collagen, a major extracellular matrix (ECM) © 2013 American Chemical Society
Received: October 8, 2013 Revised: December 17, 2013 Published: December 18, 2013 206
dx.doi.org/10.1021/bm4015039 | Biomacromolecules 2014, 15, 206−218
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functional hepatocytes for liver tissue engineering, cell therapy, and transplantation. Decellularization of tissues or organs has been investigated as a promising strategy to fabricate functional scaffolds for cell culture and transplantation.10,11 Through decellularization using detergents and enzymes, cellular components are removed from the tissues, while ECMs and some growth factor proteins are retained.10 Various ECM components including collagen, fibronectin, and laminin in decellularized matrices provide a microenvironment to cultured cells that is similar to that of native tissues and can promote survival, proliferation, and differentiation of the cells.10 Additionally, the absence of cellular components allows these matrices to serve as functional scaffolds with minimal immunogenicity for cell transplantation.12 Indeed, several types of decellularized organs derived from heart,13,14 blood vessels,15−17 heart valves,18,19 lung,20 brain,21,22 and kidney23 have been reported as functional tissue engineering scaffolds. Functional bioengineered livers for tissue engineering, organ transplantation, and drug discovery can be constructed using the decellularization process.24 Cellular components can be completely removed from the liver tissue by perfusion of detergents and nucleases, while the structural and functional features of intact liver-specific ECMs and vascular networks are well preserved.25,26 Decellularized livers can be efficiently recellularized with hepatocyte perfusion25,27 and sometimes together with endothelial cells,27 resulting in production of vascularized whole-liver constructs. The recellularized matrix supports hepatocyte survival and hepatic functions such as albumin secretion, urea synthesis, and cytochrome P450 expression at comparable levels to normal liver.25 Decellularized liver was also demonstrated to induce lineage restriction of human hepatic stem cells toward functional hepatocyte-like cells.26 Most studies have primarily focused on the application of decellularized whole-liver constructs,24−28 but the versatility of decellularized liver tissue to prepare other types of biomaterials such as coating substances or injectable hydrogels has also been reported.29,30 In this study, we report a versatile platform prepared from liver extracellular matrix (LEM) providing two functions, twodimensional (2D) substrate coating and three-dimensional (3D) injectable hydrogel, for culture and transplantation of functional hepatocytes. We provide extensive characterization of LEM coating (surface properties, surface atomic composition) and LEM hydrogel (internal structure, mechanical properties, gelation kinetics). Primary rat hepatocytes cultured on the LEM-coated substrate exhibited higher viability and improved hepatic functions compared to cells cultured on a noncoated or typical collagen type I (Col I)-coated substrate. LEM coating also enhanced the gene expression of hepatocyte markers in human adipose-derived stem cells (hADSCs). LEM hydrogel used for 3D hepatocyte culture showed higher elastic modulus compared with Col I hydrogel, which is more similar to the modulus of native liver. Structural and mechanical similarity of LEM to native liver supported the viability and functions of hepatocytes during in vitro culture. More importantly, LEM hydrogel maintained the hepatic phenotype and functions of transplanted hepatocytes in vivo. Our study demonstrates that LEM can provide biomimetic 2D and 3D microenvironments for hepatocyte survival, function, and differentiation, indicating its potential application in liver tissue engineering.
Article
EXPERIMENTAL SECTION
Preparation of LEM. The rat livers (Sprague−Dawley rats, 8week-old males, Nara Biotech, Pyungtaek, Korea) were decellularized using a modified perfusion method described in a previous study.27 In brief, rats were anesthetized with xylazine (10 mg/kg, Bayer Korea, Ansan, Korea) and ketamine (100 mg/kg, Yuhan, Seoul, Korea). A 21gauge catheter was inserted into the portal vein, and then cold distilled water was perfused through the cannulated catheter for 30 min. Subsequently, a decellularizing solution (1% (v/v) Triton-X 100 [Wako Pure Chemical Industries, Osaka, Japan] and 0.1% (v/v) ammonium hydroxide [Sigma, St. Louis, MO, U.S.A.] in distilled water) was perfused for 4 h to remove cellular components in the liver. Finally, distilled water was perfused to wash out the decellularizing solution in the liver matrix. The resultant LEM was lyophilized and stored at 4 °C until use. For further applications, the lyophilized LEM was chopped and solubilized with 10% (w/w) pepsin (Sigma) in 0.1 M HCl with stirring for 48 h at room temperature. The final concentration of LEM solution was adjusted to 40 mg/mL and further diluted with 0.1 M HCl if necessary. Characterization of LEM. Decellularization of the liver was confirmed by quantification of DNA content and histological analysis. The DNA content in LEM and native liver tissue was determined by isolating DNA from each sample using a DNA extraction kit (Bioneer, Daejeon, Korea) and measuring the absorbance at 260 nm. The content of glycosaminoglycans (GAGs) in LEM and native liver was quantified using 1,9-dimethyl methylene blue (Sigma) dye solution as previously described.31 The standard curve for determining the GAG content was generated by using serial dilutions of 20 μg/mL chondroitin sulfate A (Sigma). For histological analysis, native liver and decellularized liver were embedded in OCT cryo-compound (Leica Microsystems, Benshieim, Germany), and the specimens were then sliced into 6 μm sections. The sectioned samples were stained with hematoxylin and eosin (H&E) to check for removal of cellular components. To confirm ECM preservation in the decellularized liver, the tissue sections were immunofluorescently stained with primary antibodies against Col I (1:100 dilution, Calbiochem, San Diego, CA, U.S.A.), laminin (1:100 dilution, Abcam, Cambridge, U.K.), and fibronectin (1:100 dilution, Abcam). The stained sections were visualized with Alexa 647-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, U.S.A.) and counterstained with 4′,6-diamidino-2phenylindole (DAPI, Vector Laboratories, Burlingame, CA, U.S.A.) for nuclear staining. Mass Spectrometry Analysis for Determining Biochemical Composition of LEM. The proteins and peptides preserved in LEM were identified with mass spectrometry. The LEM solution (10 mg/ mL) was ultracentrifuged using centrifugal filter units (Millipore, Billerica, MA, U.S.A.) to remove undigested tissue debris. Liquid chromatography−tandem mass spectrometry (LC-MS/MS) analysis was performed using an integrated system composed of nano LC (Tempo nano LC system, MDS SCIEX, Ontario, Canada) and a Quadrupole-TOF MS/MS spectrometer (QStar Elite, Applied Biosystems, Foster City, CA, U.S.A.) equipped with a nanoelectrospray ionization source at an ion spray voltage of 2.3 keV. Solutions containing pepsin-digested protein fragments were injected into the nano LC-MS/MS system and then separated on a Zorbax 300SB-C18 capillary column (Agilent Technologies, Palo Alto, CA, U.S.A.). The loaded samples were eluted with a 2−35% gradient of solvent B for 30 min, then a 35−90% gradient for 10 min, followed by 90% solvent B for 5 min, and finally 5% solvent B for 15 min at a flow rate of 300 nL/minute. Solvent A consisted of water/acetonitrile (98/ 2 [v/v]) and 0.1% formic acid, and solvent B consisted of water/ acetonitrile (2/98 [v/v]) and 0.1% formic acid. Data acquisition and processing were performed with Analyst QS 2.0 software (Applied Biosystems). Generated MS/MS data were compared to the Rattus databases from NCBI using ProteinPilot software (Applied Biosystems and MDS SCIEX). LEM Coating. For culture plate coating, LEM solution was diluted with 0.2 M AcOH (Sigma) to a final concentration of 0.2 mg/mL or 2 mg/mL. Col I (BD Biosciences, Bedford, MA, U.S.A.) solution (0.2 207
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mg/mL) was used as a control coating substance. The solutions were poured onto polystyrene (PS) plates and incubated for 1 h at 37 °C. The coated plates were washed three times with phosphate-buffered saline (PBS, Sigma). To visualize deposition of Col I, a major ECM protein in liver, the coated plates were immunofluorescently stained with Col I antibodies (Calbiochem) and sequentially with Alexa 647conjugated secondary antibodies (Invitrogen). The water contact angle was measured using a Phoenix 300 goniometer (Surface Electro Optics Co., Suwon, Korea) with a constant volume (15 μL) of water droplets to confirm the change in surface properties after coating. X-ray photoelectron spectrometry (XPS, ESCALAB 220i-XL, VG Scientific Instrument, East Grinstead, U.K.) was used to examine the atomic chemical composition of the coated surfaces. LEM Hydrogel Formation. To construct 3D hydrogels, LEM or Col I solution was mixed with 10% (v/v) 10× PBS in distilled water. The pH of the solution was adjusted to 7.4 with 0.5 N NaOH, and the solution was then incubated for 30 min at 37 °C. The final concentration of LEM in the hydrogels was adjusted to 10 mg/mL or 20 mg/mL. The internal structure of the hydrogels was examined with a field emission scanning electron microscope (FE-SEM, JEOL7001F, Tokyo, Japan). The mechanical properties of the hydrogels were analyzed with a rotating rheometer (Bohlin Advanced Rheometer, Malvern Instruments, Worcestershire, U.K.). In the frequency sweep mode, the hydrogels were placed on a 20 mm parallel plate, and the storage modulus (G′) and loss modulus (G″) of the hydrogel samples were recorded at 1% strain within predetermined frequency ranges (0.079−6.33 Hz). To monitor gelation kinetics of hydrogels in the time sweep mode, the pregel mixture was placed on the plate at 10 °C, and the temperature was then rapidly increased to 37 °C to complete the gelation process. The storage modulus (G′) and loss modulus (G″) of the hydrogels were recorded at a constant frequency (0.159 Hz) and stress (1 Pa). The crossing point of G′ and G″ was considered as the gelation time.32 Culture of Primary Hepatocytes. Primary hepatocytes were isolated from Sprague−Dawley rats (8-week-old males, Nara Biotech) or Balb/c mice (8-week-old females, Nara Biotech) using a protocol modified from the collagenase perfusion method.33 The viability of harvested hepatocytes was examined with the trypan blue exclusion test. More than 80% of the harvested cells were alive. Freshly isolated primary hepatocytes were seeded (1.25 × 105 cells/cm2) on plates coated with LEM (0.2 mg/mL) or Col I (0.2 mg/mL) and cultured in Hepatocyte Culture Medium (HCM, Lonza, Walkersville, MD, U.S.A.) in humidified air with 5% CO2 at 37 °C. For 3D culture, fresh hepatocytes were encapsulated (7.5 × 105 cells/100 μL of hydrogel) in LEM or Col I hydrogel. The pregel solution with hepatocytes was fully cross-linked by incubating at 37 °C for 40 min and then carefully washed with PBS and immersed in HCM. The viability of hepatocytes cultured on the coated substrates or in the hydrogels was examined using a Live/Dead viability/cytotoxicity kit (Invitrogen). In this kit, calcein stains the cytoplasm of viable cells green, and an ethidium homodimer stains the nuclei of nonviable cells red.32 Fluorescent signals were observed under a fluorescent microscope (IX71, Olympus, Shinjuku, Tokyo, Japan). The cell viability was determined by calculating the ratio of live cells to the total cell populations. The adhesion and viability of hepatocytes on the LEM-coated substrates were also compared with those on the substrates coated with Matrigel (BD Biosciences). Analysis of Hepatocyte Functions. Albumin secretion and urea synthesis of hepatocytes were analyzed to evaluate the functions of cultured hepatocytes. During the 2D and 3D culture, culture medium was retrieved every day and stored at −80 °C until analysis. The amount of albumin in the medium was measured using an enzymelinked immunosorbent assay (ELISA, Koma Biotech Inc., Seoul, Korea). The urea concentration in the medium samples was determined using a Stanbio urea nitrogen (BUN) kit (Stanbio Laboratory, Boerne, TX, U.S.A.) according to the manufacturer’s instructions. In Vitro Hepatic Differentiation of hADSCs. hADSCs were purchased from Invitrogen and expanded in basal medium: low-
glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Gaithersburg, MD, U.S.A.) supplemented with sodium bicarbonate (3.7 g/L), penicillin (100 U/mL), streptomycin (100 mg/mL), and 10% (v/v) fetal bovine serum (Gibco BRL). When seeded hADSCs reached more than 90% confluence, hepatic differentiation of hADSCs was induced on culture substrates coated with LEM solutions at two different concentrations (1 and 2 mg/mL) using a modified three-step protocol:34 (1) mesoderm induction: cells were cultured in basal medium supplemented with activin A (50 ng/mL, PeproTech, Rocky Hill, NJ, U.S.A.) and fibroblast growth factor-4 (FGF-4, 10 ng/mL, PeproTech) for the first 3 days; (2) hepatoblast induction: cells were cultured in basal medium supplemented with FGF-4 (30 ng/mL), hepatocyte growth factor (HGF, 50 ng/mL, PeproTech), oncostatin M (OSM, 20 ng/mL, PeproTech), and nicotinamide (4.9 mmol/L, Sigma) for the next 7 days; and (3) hepatocyte induction: cells were cultured in basal medium supplemented with HGF (40 ng/mL), epidermal growth factor (20 ng/mL, PeproTech), OSM (10 ng/mL), and dexamethasone (10−7 mol/L, Sigma) for 11 days. Quantitative Real-Time Polymerase Chain Reaction (qRTPCR). After 21 days of differentiation, the gene expression of hepatic markers in differentiated hADSCs was examined with qRT-PCR. Total RNA was extracted from each sample (n = 3 per group) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, U.S.A.). Reverse transcription was carried out using the Takara PrimeScript II first strand cDNA Synthesis kit (Takara, Shiga, Japan). qRT-PCR was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) with TaqMan Fast Universal PCR Master Mix (Applied Biosystems). Gene expression of each marker was quantified using TaqMan Gene Expression Assays (Applied Biosystems) for human albumin (Hs00910225_m1), human HGF (Hs00300159_m1), human α1-antitrypsin (A1AT) (Hs00165475_m1), human α-fetoprotein (AFP) (Hs00173490_m1), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs02758991_g1). The level of gene expression was determined with the comparative Ct method in which the target genes were normalized to the endogeneous reference (GAPDH).35 The relative gene expression of each marker in hADSCs cultured on LEM- or Col I-coated substrates was normalized to that in hADSCs cultured on the noncoated substrate. Hepatocyte Transplantation. LEM or Col I hydrogel was used for hepatocyte transplantation into ectopic sites of athymic mice (Balb/c nu, 8-week-old females, Nara Biotech). Animal experiments for cell transplantation have been approved and performed in compliance with the Institutional Animal Care and Use Committee at the Yonsei Laboratory Animal Research Center (IACUC protocol number: 2010−0049). Primary rat hepatocytes (7.5 × 105 cells/100 μL of hydrogel) were mixed with pregel solution, and mice were subcutaneously injected into both sides of their backs using 31-gauge syringes. Seven days after injection, the hydrogels were harvested with adjacent skin tissues, fixed in OCT cryo-compound, and sectioned for histological analysis (n = 4). The weight of hydrogel constructs was also measured at the time of sample harvest (n = 4). The sectioned tissues were stained with H&E or immunofluorescently stained using rat albumin antibodies (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) and Alexa 647-conjugated secondary antibodies (Invitrogen). To examine the level of hepatic gene expression in hepatocytes transplanted in hydrogels, mouse hepatocytes were transplanted into the subcutaneous space of mice using LEM or Col I hydrogel. Hepatic gene expression was quantified using qRT-PCR with TaqMan Gene Expression Assays (Applied Biosystems) for mouse albumin (Mm00802090_m1), mouse hepatocyte nuclear factor-4α (HNF-4α; Mm00433964_m1), and mouse GAPDH (Mm99999915_g1). The relative gene expression of each marker in hepatocytes transplanted in LEM hydrogel was normalized to that in hepatocytes transplanted in Col I hydrogel. Statistical Analysis. All quantitative data are given as mean ± standard deviation. Statistical significance was determined using an unpaired Student’s t test with Sigma-Plot software (Systat Software Inc., Chicago, IL, U.S.A.). A p value
