Abstract
Liver transplantation is currently the most efficacious treatment for end-stage liver diseases. However, one main problem with liver transplantation is the limited number of donor organs that are available. Therefore, liver tissue engineering based on cell transplantation that combines materials to mimic the liver is under investigation with the goal of restoring normal liver functions. Tissue engineering aims to mimic the interactions among cells with a scaffold. Particular materials or a matrix serve as a scaffold and provide a three-dimensional environment for cell proliferation and interaction. Moreover, the scaffold plays a role in regulating cell maturation and function via these interactions. In cultures of hepatic lineage cells, regulation of cell proliferation and specific function using biocompatible synthetic, biodegradable bioderived matrices, protein-coated materials, surface-modified nanofibers, and decellularized biomatrix has been demonstrated. Furthermore, beneficial effects of addition of growth factor cocktails to a flow bioreactor or coculture system on cell viability and function have been observed. In addition, a system for growing stem cells, liver progenitor cells, and primary hepatocytes for transplantation into animal models was developed, which produces hepatic lineage cells that are functional and that show long-term proliferation following transplantation. The major limitation of cells proliferated with matrix-based transplantation systems is the high initial cell loss and dysfunction, which may be due to the absence of blood flow and the changes in nutrients. Thus, the development of vascular-like scaffold structures, the formation of functional bile ducts, and the maintenance of complex metabolic functions remain as major problems in hepatic tissue engineering and will need to be addressed to enable further advances toward clinical applications.
Introduction
The main causes of liver disease include alcoholism, drug abuse, hepatitis B or C virus infection, cholestasis, and metabolic syndrome. Liver failure is associated with rapidly progressive multiorgan failure and devastating complications. Chronic end-stage liver disease and acute liver failure are associated with high mortality because of loss of liver functions (6,91).
Among the currently available therapies, orthotopic liver transplantation is an accepted and effective way to treat hepatic failure. However, it cannot be used in many patients due to limited organ availability (6,93,108). Transplantation of functional cells including hepatocytes and hepatic lineage stem or progenitor cells may offer an alternative cell-based therapy to orthotopic liver transplantation for the treatment of hepatic failure and hereditary liver disease (26,55,64,74,82).
Cell Sources
The liver consists of many cell types with multiple metabolic functions. Hepatocytes are one of the main cell types in the organ. Hepatocytes maintained in culture provide an attractive model system for the study of liver function (106). Multiple papers have been published reporting the use of primary hepatocytes for tissue engineering. Primary hepatocytes are usually derived from a two- or three-collagenase method for treating the liver (17,86–88). However, their use is limited because of a shortage of hepatocytes that can be isolated and cultured. They are mature cells with short telomeres (124). Other sources of hepatocytes must be found. Table 1 shows the current state of cell systems that can be used in liver tissue engineering to replace hepatocytes.
Types of Stem Cells Currently Used for Liver Tissue Engineering
FLCs, fetal liver cells; AFP, α-fetoprotein; CK, cytokeratin; HNF, hepatocyte nuclear factor; CYP, cytochrome P450; VEGF, vascular endothelial growth factor; IL, interleukin; HGF, hepatocyte growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; MSC, mesenchymal stem cell; ECM, extracellular matrix; SIS, small intestinal submucosa; OsM, oncostatin M; Dex, dexamethasone; ITS, insulin/transferrin/selenium; AS-2P, ascorbic acid-2-phosphate; TO, tryptophan 2,3-dioxygenase; AAT, α1-antitrypsin; TAT, tyrosine aminotransferase; G6PC, glucose-6-phosphatase; CXCR4, chemokine (C-X-C motif) receptor 4; EpCAM, epithelial cell adhesion molecule; GGT1, gamma glutamyl transpeptidase; AE2, anion exchange 2; ASBT, apical sodium-dependent bile acid transporter; ASMA, α-smooth muscle actin; SR, secretin receptor; HDL, high density lipoprotein; T3, tri-iodothyronine; Flk 1, fetal liver kinase; PDGF, platelet-derived growth factor; ITO, hepatic stellate cells; PLGA, poly-lactide-co-glycolide; ICG, indocyanine green; BMP, bone morphogenetic protein; Sox17, SRY (sex-determining region Y)-box 17; Oct-4, octamer-binding transcription factor 4.
Additional choices for sources of hepatocytes include the early fetal liver at around embryonic days 12–16, which contains two populations of hepatic cells: fetal hepatic stem cells and hepatic progenitor cells (hepatoblasts), which are δ-like 1 homolog positive (Dlk-1+) and thymocyte differentiation antigen 1 negative (Thy-1-), respectively (98). Liver stem/progenitor cells (oval cells) express oval cell marker 6 (OV6), cytokeratin-19 (CK19), and CK7 (9) and share expression of some markers with hematopoietic cells, such as Thy-1, cluster of differentiation 34 (CD34), and stem cell antigen-1 (Sca-1) (15,100). In addition, pancreatic cells can transdifferentiate (convert/reprogram) into hepatocytes (90).
Adult stem cells such as mesenchymal stem cells (MSCs) typically give rise to many types of mesodermal tissues, such as bone, cartilage, smooth muscle, and fat (77). Recently, these cells have been shown to not only differentiate into hepatic lineage cells but to also functionally alleviate chemically induced liver fibrosis in rats (13,52), showing potential for use in liver therapy. Schwartz et al. found that with a seeding density less than 1.25×104 cells/cm2, no hepatic differentiation of human MSCs in monolayers on fibronectin, collagen, or Matrigel occurs (85).
Embryonic stem (ES) cells are considered a part of the extrahepatic compartment. Due to their pluripotent capability, ES cell-derived cells may be a potential future source of hepatocytes (63). In vitro, they can potentially differentiate into functional hepatocyte-like cells under chemically defined conditions elucidated from embryonic developmental biology (4). Several molecules can activate signaling via the activin A receptor and Wnt3a pathways (30), these include activin A, fibroblast growth factors (bFGF, aFGF, FGF10), bone morphogenetic proteins (BMP2, BMP4), retinoic acid, hepatocyte growth factor (HGF), epidermal growth factor (EGF), dexamethasone, insulin–transferrin–selenium (ITS), oncostatin M, dimethyl sulfoxide, sodium butyrate, etc. (10,45,94). Injection of functional hepatocyte-like cells (HLCs) derived from human ES cells into the spleen of acutely injured immune-compromised mice shows that functional HLCs persist and remain engrafted for up to 3 months after transplantation (71). Touboul et al. also found that green fluorescent protein-expressing lentivector-transfected differentiated cells (lenti-GFP hES cells) engrafted into immunodeficient urokinase-type plasminogen activator transgenic mouse liver [developed in 1990 by Heckel et al. (31)] grow and maintain functional expression for at least 8 weeks (104).
In 2006, Takahashi and Yamanaka first demonstrated induction of pluripotent stem (iPS) cells from mouse embryonic or adult fibroblasts by introducing four factors: octamer-binding transcription factor 3/4 (Oct3/4), sex-determining region Y box 2 (Sox2), myelocytomatosis viral oncogene homolog (c-Myc), and Krüppel-like factor 4 (Klf4) (102), suggesting great promise for iPS cells in regenerative medicine (2,53).
Biomaterials for Tissue Engineering
For replacement or restoration of hepatic tissues that have been damaged by disease or injury, tissue engineering has become more important. Tissue engineering requires three important components: cells, scaffold materials, and growth factors.
Recent findings show that stem cell differentiation toward liver lineages is promoted by three-dimensional (3D) dynamic perfusion culture conditions, which more closely resemble the in vivo environment regarding correct cell morphology, cellular environment, gene expression, and biological behavior of the cells (3,58). The function and therapeutic potential of transplantable cells for liver therapy can be directed in an appropriate manner and may involve presenting the cells with the appropriate extracellular matrix (ECM) substrates that enhance the functions, engraftment, and survival of the cells. Creating 3D microenvironments that better mimic normal in vivo conditions and encapsulating the cells to partially isolate them from the potentially hostile in vivo microenvironment in the lesion are strategies that may enhance transplantation success (63). Other physical parameters, such as oxygen tension, can also affect stem cell proliferation and differentiation in particular cases (1). The substrate material is also an important factor for long-term cryopreservation of hepatocytes. Lu et al. found that polyvinyl alcohol is an appropriate and promising substrate for culturing hepatocytes that have been cryopreserved for more than 4 years without resulting in the loss of liver-specific functions (56). Another group showed that hepatocytes at room temperature can be preserved for more than 10 days in a 3D culture system in the presence of 0.25 mg/ml epigallocatechin-3-gallate [EGCG] (59). Recent concepts involved in liver therapy are schematically shown in Figure 1.
Diagram of the desirable (ideal) pattern of liver regenerative medicine, which includes tissue engineering, bioartificial liver technology, and cell transplantation therapy.
Distinct cellular behaviors are observed in 3D culture systems that are not observed in standard monolayer two-dimensional (2D) cultures, indicating that 3D culture conditions may mimic the in vivo environment more closely than 2D culture (48,49). Moreover, 3D culture may benefit hepatocyte maturation and function because a 3D culture provides an environment that is more like that in the human body and that enhances cell–cell interactions. However, with 3D culture methods, many complex parameters must be considered, the most important of which is the choice of materials. Optimal materials for biomedical therapy must be biocompatible and biodegradable. We will review current papers on this topic as shown in Table 2.
Materials for Liver Application
PEI, polyetherimide; PAA, polyacrylic acid; PEX or XLPE, cross-linked polyethylene; PE, polyethylene; PET or PETE, polyethylene terephthalate; PPE, polyphenyl ether; PVC, polyvinyl chloride; PVDC, polyvinylidene chloride; PLA, polylactic acid; PP, polypropylene; PB, polybutylene; PBT, polybutylene terephthalate; PA, polyamide; PI, polyimide; polycarbonate; PEI, polyetherimide; PTFE, polytetrafluoroethylene; PS, polystyrene; PU, polyurethane; PEs, polyester; ABS, acrylonitrile butadiene styrene; PMMA, poly (methyl methacrylate); POM, polyoxymethylene; PES, polysulfone; EVA, ethylene vinyl acetate; SAM, styrene maleic anhydride; PEEK, polyetheretherketone; PLGA, poly (lactic-co-glycolic acid); EGCG, epigallocatechin-3-gallate.
Chemically Synthesized Materials
Poly (d,l-Lactic-co-Glycolic Acid) (PLGA)
Hepatic cells can be cultured for use in tissue engineering, drug screening, or to treat the damaged liver, purposes that require cells that are physiologically functional in vivo. For this reason, biodegradable polymers such as poly-dimethylsiloxane (PDMS), poly (D,L-lactic acid) (PLLA), PLGA, and polyurethane-acrylate (PUA) are usually used to create a scaffold that provides a microenvironment similar to the in vivo environment, including a 3D ECM, a high supply capacity of oxygen and nutrients, and high cell density. Previous studies have shown survival of hepatocytes (5.3 to 7.8 × 107 cells/polymer) cocultured with nonparenchymal liver cells (2.7 to 3 × 107 cells/polymer) on synthetic biodegradable polymer (PLGA, 85 Lactic:15 glycolic acid, 100,000 MW) discs under continuous flow conditions (1.1 or 1.4 ml/min) in vitro and have demonstrated the capacity of hepatocytes to attach to these devices at high densities, to survive after attaching to these polymers in static and flow conditions, and to synthesize albumin (39). Porous foam consisting of amorphous 50 Lactic:50 glycolic acid PLGA (75,000 MW) shows microporosity on the surface and significantly improves primary hepatocyte attachment and viability. Foam with subcellular-sized voids (~3 μm) induces kinetics of 2D hepatocyte reorganization, but 3D aggregation is limited. However, larger subcellular-sized voids (~67 μm) restrict hepatocyte motility, thereby promoting the kinetics of 3D aggregation. Subcellular-sized voids between these sizes (~17 μm) promote both 2D and 3D reorganization kinetics. In addition, the topography of nonphysiological polymer scaffolds is also a powerful variable in the microengineering of hepatospecific activity of the resulting tissue analogs (79). Recently, another group used 3% type 1 collagen-coated PLGA as a 3D bio-scaffold and showed improvement in the differentiation of F344 rat bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells that express the hepatocyte-specific markers albumin, α-fetoprotein, cytokeratin 18, hepatocyte nuclear factor 4α, and cytochrome P450 at the mRNA and protein levels within 3 weeks (50).
Poly-l-Lactic Acid (PLLA)
Sakai and colleagues investigated C57Bl/6CrALc embryonic day 14.5 fetal mouse liver cells (2.0 × 104 cells/cm2) or embryonic day 17 fetal Wistar rat liver cells (4.0 × 104 cells/cm2) that were cultured in 0.03% type 1 collagen-coated 3D PLLA (300,000 MW) scaffolds (2 mm thick; 10 mm diameter, 0.16 cm3 volume) in the presence of HGF, FGF1, FGF4, sodium butyrate, nicotinamide, dimethyl sulfoxide, and oncostatin M and found greater hepatic function that was well maintained during the 2–4 weeks of culture. Implantation of cells grown in this manner into the peritoneal cavity of 70% hepatectomized mice showed a remarkably higher presence of albumin-positive engrafted cells 15 days after transplantation (29,35).
Poly-Dimethylsiloxane (PDMS)
For scaffolds containing PDMS, HepG2 cells at high density were cultured in low O2 (average maximal cellular oxygen consumption rate of 3.4 × 10−17 mol/s/cell), and the oxygen transfer limit factor was discussed (76). Culture conditions for optimum perfusion involve the design of the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5 × 107 cells/ml) cell culture. Maximum metabolic rate densities for this scaffold are 6.04 × 10−3 mol/s/m3 for O2 at a flow rate of 0.71 ml/min and 1.91 × 10−2 mol/s/m3 for glucose at a flow rate of 0.35 ml/min in the scaffold (20 mm width x 35 mm length) as determined with computational fluid dynamics analysis (75).
Polyurethane Foam (PUF)
A hollow-fiber, poly-amino-urethane-coated, unwoven, polytetrafluoroethylene (PTFE) fabric-type (PAU-coated PTFE) bioartificial liver (BAL) module was developed. Mouse ES cells cultured in such materials in the presence of the hepatic-like cell differentiation factors FGF2, HGF, dimethyl sulfoxide, and dexamethasone differentiate into hepatocyte-like cells (95). When mouse ES cells are cultured in a hybrid artificial liver using the PUF/hepatocyte spheroid method, the cells proliferated by 20 days achieved a high cell density (about 1 × 108 cells/cm3 PUF) and expressed endodermal-specific genes (57). Recently, another group found that using 0.25 mg/ml EGCG not only facilitates various bioactivities but also ensures the function of porcine hepatocytes at room temperature, especially in a 3D culture system, and may be beneficial for BAL construction (59).
Polyvinyl Fluoride (PVF)
PVF resin can be used in BAL construction as a 3D porous scaffold in packed-bed reactor perfusion systems and can immobilize mouse and pig fetal liver cells (60).
Polycaprolactone (PCL)
Hutmacher and coworkers have reported fused deposition modeling technology by melting the thermoplastic biodegradable polymer PCL (34). To evaluate its biocompatibility, human hepatoma HepG2 cells were seeded into the PCL, and 80% (w/w) NaCl salt particles that serve as a porogen hybrid scaffold were applied using a selective laser sintering process. This method produces high (89%) porosity with a pore size of 100–200 μm in the 3D flow channel network. Using avidin–biotin binding, cells cultured in a perfusion system for 9 days grow and maintain their functions, showing promise for the in vitro development of large, implantable liver tissues (33).
Materials Derived from Natural Components
Alginate Beads
Alginate from the cell walls of brown algae is a natural polysaccharide that has recently been used in liver-assist devices. Encapsulation of HepG2 or C3A cells maintains the function and growth of the cells (40,44). Extracorporeal perfusion system expansion (78) and the fluidized bed BAL culture system (40) increase proliferation rates in a microgravity environment compared with static cultures (16). In addition, long-term maintenance of liver cells may have potential for use in drug screening (44).
Chitosan
Primary rat hepatocytes (1.5 × 106 cells/ml) cultured in gelatin/chitosan (10:1) hybrid material with open channels made by the cell assembly machinery show tissue-like morphology and retain expression of glutamate–oxaloacetate transaminase, albumin, urea, glucose, and creatinine, as well as triglyceride secretion function (119). Recently, the use of electrospinning technology to fabricate nanofibrous scaffolds showed that natural material, such as galactosylated chitosan (GC), can be made into nanofibers. Rat hepatocytes form immobile, 3D, flat aggregates and exhibit superior cell bioactivity with higher levels of liver-specific albumin secretion, urea synthesis, and cytochrome P450 enzyme expression when grown on GC film (25). These developing techniques may provide an excellent substrate for primary hepatocyte cultures for BAL or tissue engineering applications.
Poly-Terephthalate (PET) Film
3D monolayer culture on PET film was developed with the strategy of surface modification such as ECM proteins coated on gelatin, collagen, laminin, fibronectin, or conjugated to cell adhesion peptides, such as Arg-Gly-Asp (RGD), Tyr-Ile-Gly-Ser-Arg (YIGSR) (11), Gly-Arg-Gly-Asp-Ser (GRGDS) (20), or galactose ligands through the galactose asialoglycoprotein receptor interaction to synergistically enhance hepatocyte adhesion and function (19). These strategies may be useful for drug-screening platforms (123) and BAL-assist devices (32).
Hydrogels
Fibrin gels are widely used, injectable scaffolds that are used for cell transplantation due to their biodegradability and support of cell infiltration and proliferation. Primary human fetal liver progenitor cells cocultured with endothelial cells in fibrin gels form vascular structures and show increased proliferation. This technique is a novel coculture system that may be useful for the development of engineered liver tissue applications (116). A biodegradable material composed of hydrogel-inverted colloidal crystal scaffolds was developed to preserve the viability and morphology of hepatocyte spheroids, allowing maintenance of differentiated structures and functions (41,46). When compared to hepatocytes grown in 2D culture, spheroids maintain higher viability and better functions. This technology was developed for use in toxicology testing systems in vitro, but it can be further modified for 3D drug-screening systems and may be applied to accurately reflect the actual toxicity of nanoparticles and other nanostructures in the body (46). Other groups have used polyethylene glycol hydrogels to encapsulate hepatic cells for assessment of hepatocellular functions. One group has explored the behavior of bipotential mouse embryonic liver cells and hepatic cells as a model for hepatocyte differentiation and maturation (106). These cells can be efficiently infected with hepatitis C virus, and progeny infectious virus can be recovered from the medium supernatant of the hydrogels (14). Another use of the polyethylene glycol hydrogel adhesive RGD peptide sequence is to ligate α5β1 integrins in cocultured hepatocytes for fabrication of a functional 3D hepatic construct in a continuous flow bioreactor for 12 days, which produces higher albumin and urea secretion (54). These techniques may be useful for tissue-engineered, implantable liver systems.
Matrigel
Recently, a paper was published comparing porcine liver-derived ECM and matrigel for the ability to support human hepatocyte maintenance and function in vitro (87). When the immortalized but nontransformed human hepatocyte cell line QSG-7701 is cultured on 3D matrigel, the cells form a tissue-like structure from a polarized acinar structure and reacquire liver-specific functions, providing an ex vivo model to study liver-specific function and tumorigenesis (122). Another group showed that the combination of particular growth factors, such as HGF and EGF, benefits aggregated growth (88).
Collagen-Based Scaffold
Collagen is a natural ECM polymer that is widely used as a scaffold for hepatocyte transplantation. This 3D cell culture model offers a unique opportunity to maintain cells in a differentiated state and to maintain the differentiated morphology of hepatocytes and suppression of α-fetoprotein synthesis in vitro (3,42). Hepatocytes grown in a 3D collagen matrix culture produce glucose at 240–290 mg/106 cells for up to 24 h compared with traditional culture conditions in which glucose is produced at less than 50 mg/106 cells. Also, higher expression of phosphoenolpyruvate carboxykinase and CCAAT/ enhancer-binding protein a in the 3D system preserves the metabolic function of hepatocytes and can be used as an in vitro model for studying hepatocyte glucose production and gluconeogenesis (112). Bile acid homeostasis is significantly higher in stable collagen sandwich cultures in vivo, indicating substantial deviation from the critical physiological behavior of rat-specific bile acid pathways (18). In addition, a 3D CYP450 metabolism cellular microarray chip platform has been developed (47). These trends demonstrate that the 3D collagen scaffold provides a unique platform to study hepatic metabolism. The collagen sandwich primary hepatocyte culture system preserves urea production, cytochrome P450 activity of mature hepatocytes for 14 days (123), and phosphorylation of HGF receptors and EGF receptors (22) for drug-screening platforms. Reference gene levels show that the novel target genes E2F transcription factor seven and interleukin 11 receptor a subunit are potential toxicity biomarkers for toxicology application for acetaminophen treatment (27).
Nanomaterials
As shown in Table 3, most nanomaterials used for liver-related research are nanofibers. Currently, three main strategies for liver failure therapy are under investigation. These include liver tissue engineering transplantation, extracorporeal BAL, and extracorporeal liver-assisted devices (61). We will focus on liver tissue engineering transplantation. Tissue engineering was developed using materials for 2 × 105 primary rat hepatocytes cultured on collagen-coated nanofibers for 1 week for pharmaceutical toxicology research. Cells cultured on this type of material secrete albumin and store glycogen (8). Another group used 0.5% (v/v) self-assembling peptide nanofiber (SAPNF) hydrogel as an ECM protein for culturing primary porcine hepatocytes that were extracted with the four-step enzyme perfusion method (43) and compared the cells with those grown on a collagen-based plate. Results showed that different cell spheroid structures form within 7 days when cells are grown on SAPNF hydrogel, and mature function is maintained at day 14 (65). Aggregated hepatocytes express connexin 32, a major hepatic gap junction protein that enables hepatic cell interaction, and this expression disappears in monolayer culture. In addition, expression of zona occludens-1 (ZO-1), a tight junction-associated peptide, indicates reformation of bile canaliculi and bipolar configuration. Inducibility of cytochrome isoenzymes, such as Cyp1a1, Cyp1a2, Cyp2b2, and Cyp3a1, was also found (8). In another experiment, mouse hepatocytes were cultured in conditioned medium (CM) from the human cell line CYNK-10 prepared with SAPNF and transplanted into the muscle of rodents. Results showed a therapeutic effect on extrahepatic site liver cirrhosis (60% survival rate) and 70% hepatectomy (75% survival rate for 7 days) and SAPNF/CM-hepatic tissue-engrafted mice survived for 48 ± 11 days (62). Some nanofiber scaffolds composed of PCL, collagen, and polyethersulfone were fabricated with the electrospinning technique. These 3D materials were used to help differentiate human bone marrow-derived mesenchymal stem cells (hBMSCs) into hepatocytes, resulting in approximately double the efficiency seen in normal 2D culture (37). Another novel use of natural nanofiber GC scaffolds to enhance primary hepatocyte aggregation produced better bioactivity as determined by albumin secretion, urea synthesis, cytochrome P450 enzyme activity, and mechanical stability than normal GC film culture (25). For this application, the ultra-web nanofibers are composed of two kinds of polyamide polymers, A (C28O4N4H47) n and B (C28O4.4N4H47) n , which are cross-linked in the presence of an acid catalyst (84). Human ES cells (hESCs) show enhanced function when grown on these nanofibers (24), and in vivo carbon tetrachlorideinduced liver fibrosis rat models transplanted with hBMSCs differentiated on nanofibers show functional recovery, decreased liver fibrosis when engrafted in the recipient liver, differentiation of functional hepatocytes (albumin+), enhanced serum albumin levels, homing of transplanted cells, and functional engraftment (73). More recently, self-assembling peptides have proved to be an excellent scaffold for cell culture, including those comprising the signaling sequence GRGDSP (RGD) from collagen, YIGSR (YIG) from laminin, TAGSCLRKFSTM (TAG) from heparin, and RAD-16I from hydrogel. In particular, RAD-16I with the PTFE novel bioengineering platform shows better improvement and maintains expression of albumin, CYP3A2, and hepatocyte nuclear factor 4α, similar to fresh hepatocytes, for as long as 1 week compared to the traditional sandwich culture method (113). Moreover, development of a HepG2 hepatocellular carcinoma in vitro model may be a powerful tool for use in cancer research and antitumor drug development (114).
Nanomaterials for Liver Cell Culture
CCl4, carbon tetrachloride; BSA, bovine serum albumin; CK, cytokeratin; FOXA2, forkhead box protein A2; CX32, gap junction β-1 protein; KOSR, knockout serum replacement; NEAA, nonessential amino acids; TO, tryptophan 2,3-dioxygenase; CXCR4, chemokine (C-X-C motif) receptor 4; C/EBPβ, the transcription factor CCAAT/enhancer-binding protein β; LDL, low-density lipoprotein; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ZO-1, tight junction protein; LDH, lactate dehydrogenase; CM, conditioned media; SCID mice, severe combined immunodeficiency mice.
Decellularized Liver Matrix Culture
A decellularized organ scaffold with renewed hepatic cells may provide another therapy choice requiring tissue engineering in the future. In 2004, Lin et al. assessed porcine liver-derived biomatrix for rat primary hepatocyte culture. Cells were maintained for up to 45 days, and liver-specific functions, such as albumin synthesis, urea production, and P450 IA1 activity, were better than functions of cells grown on adsorbed collagen cultures (51). Uygun et al. published a paper in Nature Medicine (107) using a similar concept of a decellularized liver matrix and showed retention of intact lobular structures and vascular beds. They observed not only maintenance of normal liver-like function in vitro but also hepatocyte survival and function with minimal ischemic damage in vivo. Moreover, Schanz and coworkers developed the biological vascularized scaffold (BioVaSc®), which is generated from a decellularized porcine small bowel segment with preserved tubular structures of the capillary network within the collagen matrix, showing that a functional vascular network can help grow model tissues for study (83).
Application of Biomaterials in Liver Therapy
Tissue engineering involving scaffold materials, cells, ECM, and protein factors has been proposed for several years. Many authors have claimed that the benefits of 3D culture, such as ES cell differentiation into hepatocytes in vitro, include better functions and more mature expression than cells cultured in normal 2D culture systems (3). In addition, human fetal or primary hepatocytes grown on biomatrix scaffolds form a 3D vessel-like network (125). When transplanted into severe combined immunodeficient mice, the matrices continue to express specific human proteins in defined, differentiated structures and appear to recruit and anastomose with the host vasculature (49). This approach provides a unique culture system for addressing questions of cell and developmental biology and provides a potential mechanism for creating viable human tissue structures for therapeutic applications (49,99). The use of hESC-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel BAL devices and predictive drug toxicity assays (89). Compared to the 2D culture of cell monolayers, 3D models more closely mimic native tissues because the cellular microenvironment established in the 3D models often plays a significant role in disease progression and cellular responses to drugs (21).
Artificial Liver
Several reviews describe the overall status, history, critical issues, cell sources (126), general bioreactor and system designs, preclinical and clinical results of current BAL systems, and future perspectives (111). In these reviews, the liver-like structure of the BAL system (70) shows high performance, and 3D culture is superior to the 2D culture system. Regarding hepatic functions, several types of materials enhance the culture system from short term to long term in the order of microcarriers (103,115), ECM, porous matrix, microencapsulation (40), spheroids or organoids (57), and collagen sandwich. For example, the use of a 3D PDMS scaffold optimizes the geometry and allows attachment of cells at high density (up to 2 × 108 cells/ml experimentally); however, an oxygen transfer coefficient limitation was noted (76). Another paper showed that hepatocytes can be preserved in the presence of 0.25 mg/ml EGCG at room temperature, especially in a 3D culture system, which may be promising for preparation of BAL (59). Scaffold porosity was also examined by utilizing a modern microbraiding technique to interlace PET fibers onto the polysulfone hollow fibers (32). Three notable trends of the bioreactor design for BAL systems have been noted. One is that an additional channel for nutrients or oxygen supply is integrated into the bioreactor. Another is that improved hepatocyte culture techniques, such as the use of spheroids, cylindrical organoids, and sandwich culture, are applied for the long term and enhance liver functions. Finally, selection of different cells, such as stem cells, progenitor cells, fetal cells, a particular cell line, or transgenic cells, is important.
Transplantation in Vivo
Treatment of chronically hepatectomized rats with a tissue-engineered liver improves liver function and prolongs survival; the mean lifespan of the rats was extended from 16–72 h (5). These results show that the topographic properties of nanofibers enhance differentiation of HLCs from MSCs and maintain their function in long-term culture, which has implications for cell therapies. A 4-week mouse survival rate can be achieved with such cells and nanofibers (73). In vivo results show that a scaffold that supports hES-derived hepatocytes allows the cells to remain viable for at least 2 weeks, that the engineered tissue may recruit and anastomose with the host vascular system and that the differentiation pattern is induced in vitro (49). Combining nonparenchymal cell types (endothelial, stellate, and cholangiocyte cells) with hepatocytes (1:3) is critical to the formation of liver structures and may extend the potential long-term efficacy of a liver-assist device by about 7 days (97). In particular, MSCs not only stabilize primary cell functions in biomedical devices but also provide an immunomodulatory component of the therapy that directly affects systemic inflammation and tissue injury and supports the notion of liver-assist devices containing cocultures of MSCs and hepatocytes as a potential therapy for acute liver failure (117). Transplantation of fetal liver cell-loaded hyaluronic acid sponges onto mesenteric blood vessels leads to thick, liver-like tissue with blood vessels, which exhibits a remarkable therapeutic effect on the copper metabolism deficiency of Long Evans Cinnamon rats. Two weeks after being fed a conditioned diet, rats transplanted with fetal liver cell-loaded hyaluronic acid sponges showed no jaundice within 3 weeks of transplantation (36). A recellularized graft supports liver-specific function including albumin secretion, urea synthesis, and cytochrome P450 expression at comparable levels to normal liver in vitro. Recellularized liver grafts can be transplanted into rats, supporting hepatocyte survival and function with minimal ischemic damage (107). In mice, the 30-day survival rates were 70% for SAPNF-hepatocyte transplantation, 50% for collagen-hepatocyte transplantation, and 40% for hepatocyte transplantation alone. Hepatocyte culture in a true 3D network allows in vivo maintenance of differentiated functions, and once transplanted, the hepatocytes can correct acute liver failure in mice and prolong their survival. Transplantation in widely divergent species seems possible (118).
A new approach to hepatocyte transplantation has been described in which an engineered functional hepatic tissue graft is successfully implanted into the skeletal muscle of mice with acute hepatic failure where it provides clinically significant hepatic support and 7-day survival. Intramuscular hepatic tissue transplantation in mice with chronic liver failure leads to a 63-day survival (62). After an engineered hepatic tissue transplantation in mice, the 30-day survival rate is 60%, but similar data were obtained with transplants of either porcine or human hepatocytes.
Construction of functional engineered hepatic tissue using PAU-coated PTFE as a platform for hepatocyte transplantation into multiple species has been demonstrated (96). Cell injection allows repeated inoculation of a BAL with fresh cells to facilitate maintenance of a functional hepatocyte mass. Use of this device in mice with acute liver failure, which uniformly die within 4 days of inducing hepatic failure by 90% hepatectomy, results in 90% survival at 12 days (95).
Conclusion
Many excellent studies have examined stem cells, biomaterials engineering, and nanobiotechnology, and the collective results promise to revolutionize liver regenerative medicine. However, progress is limited by a lack of understanding of the specific molecular mechanisms in the microenvironment that affect hepatic lineage cells and the signaling pathways that regulate efficient differentiation of stem cells and adult tissue formation. Based on the ongoing study of biomaterials scaffolds and stem cells, a tissue decellularized scaffold combined with iPS cells for tissue engineering for cell therapy or regenerative medicine is possible. Although the in vivo use of iPS cell-seeded decellularized biomatrix has not been described, these studies are likely imminent. In conclusion, the mimic liver tissue system combined with genetic engineering has great potential as a powerful tool for efficient ex vivo stem cell expansion and maintenance of mature functions for future personalized transplantation approaches.
Footnotes
Acknowledgments
This work was partly supported by grants from the National Science Council of the Republic of China and the Gwo Xi Stem Cell Applied Technology, Co., Hsinchu, Taiwan, ROC. The authors declare no conflict of interest.