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Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets Kazuo Ohashi1,6, Takashi Yokoyama1, Masayuki Yamato2, Hiroyuki Kuge1, Hiromichi Kanehiro1, Masahiro Tsutsumi3, Toshihiro Amanuma4, Hiroo Iwata5, Joseph Yang2, Teruo Okano2 & Yoshiyuki Nakajima1 Hepatic tissue engineering using primary hepatocytes has been considered a valuable new therapeutic modality for several classes of liver diseases. Recent progress in the development of clinically feasible liver tissue engineering approaches, however, has been hampered mainly by insufficient cell-to-cell contact of the engrafted hepatocytes. We developed a method to engineer a uniformly continuous sheet of hepatic tissue using isolated primary hepatocytes cultured on temperature-responsive surfaces. Sheets of hepatic tissue transplanted into the subcutaneous space resulted in efficient engraftment to the surrounding cells, with the formation of two-dimensional hepatic tissues that stably persisted for longer than 200 d. The engineered hepatic tissues also showed several characteristics of liver-specific functionality. Additionally, when the hepatic tissue sheets were layered in vivo, three-dimensional miniature liver systems having persistent survivability could be also engineered. This technology for liver tissue engineering is simple, minimally invasive and free of potentially immunogenic biodegradable scaffolds. To meet the substantial demand and provide potential therapeutic benefits for persons suffering from liver disease, the development of new therapeutic applications is crucial. Approaches using hepatocytebased therapies have already been experimentally and clinically explored1,2 as a result of their relative simplicity and minimal invasiveness to the individual, and have triggered new interest in the bioengineering of alternative liver systems in vivo based upon the manipulation of in vitro–modified hepatocyte cultures2,3. Additionally, recent advances in methods for the efficient derivation of hepatocytes from embryonic stem cells4 provide a plausible alternative cell source for future hepatic tissue engineering approaches. Diseases that could potentially benefit from the bioengineering of new liver tissues therefore include congenital metabolic liver disorders, which would not require replacement of the entire organ. Over the past 10 years, the liver tissue engineering field has empirically developed two main approaches, involving the transplantation of suspended hepatocytes with extracellular matrix components5–9
or the use of biodegradable scaffolds to provide a platform for hepatocyte attachment3,10. As a proof-of-concept study, we have transplanted hepatocytes under the kidney capsule and successfully engineered an ectopic liver system that persisted over the long term and possessed full functional activity during liver regeneration6,7. Because of the invasive nature of this approach, however, alternative sites of transplantation—for example, manipulations of the subcutaneous space—must be considered so that repeated procedures can be easily and safely performed. The establishment of a subcutaneous transplantation site to engineer liver tissue systems would therefore be a significant development for future clinical applications. Earlier studies using scaffold-based designs to engineer tissues for transplantation have faced several drawbacks, such as inflammatory responses and fibrosis11. Here, however, we use a special cell culture dish coated with the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm)11–18, in which temperature fluctuations can mediate changes in the chemistry of the polymer coating. At 37 1C, PIPAAm is slightly hydrophobic, allowing cells to be cultured under normal conditions. A decrease in the temperature below 32 1C, however, results in rapid hydration of the polymer, leading to the spontaneous detachment of the cells as a uniform tissue sheet. As the temperature-responsive polymer is covalently immobilized onto the culture surfaces, PIPAAm remains bound to the dishes even after cell detachment. Using this technology, tissue sheets composed of oral mucosal epithelial cells have been generated and are now clinically applied for corneal reconstruction, with this approach being noted for its simplicity and favorable clinical results14. Thus, the ability to create tissues in a scaffold-independent manner should provide additional benefits, by minimizing exposure to potentially immunogenic molecules, as well as reducing the risk of inflammatory responses. RESULTS Bioengineering of functional hepatic tissue sheets We used transgenic mice (hA1AT-FVB/N) that expressed human a1antitrypsin (hA1AT) driven by the A1AT hepatocyte-specific promoter as donors for the hepatocytes in the present study. After hepatocyte isolation and purification, we cultured hA1AT-expressing mouse
1Department of Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. 2Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. 3Department of Pathology, Saiseikai Chuwa Hospital, 323 Abe, Sakurai, Nara 633-0054, Japan. 4Nihon Noyaku, Co. Ltd., 345 Oymada-cho, Kawachinagano, Osaka 586-0094, Japan. 5Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. 6Present address: Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan. Correspondence should be addressed to K.O. (
[email protected]).
Received 14 February; accepted 9 March; published online 17 June 2007; doi:10.1038/nm1576
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Figure 1 Morphological validation of the engineered hepatic tissue sheets composed of primary hepatocytes. (a) Gross appearance of the engineered hepatic tissue sheet harvested by temperature-dependent removal from a PIPAAm culture dish. (a–d) Histological analyses of the harvested hepatic tissue sheets by hematoxylin-eosin staining (b), hA1AT immunostaining (c) and albumin immunostaining (d). (e) Strong immunofluorescence was detected for fibronectin along the bottom of the hepatic sheet. (f) Transmission electron microscopy showed microvillus development (black arrowhead) on the apical surface of the tissue sheet, characteristic cell-tocell connections in terms of gap junctions (black arrow), and the presence of bile canaliculi (white arrowhead) and desmosomes (white arrows). (g) Extracellular matrix (ECM) components were deposited by the hepatocytes during culture and were found to be attached to the bottom side of the tissue sheet. (h) Histological analysis of the harvested hepatic tissue sheet composed of isolated human hepatocytes. Scale bars: 1 cm (a), 100 mm (b), 50 mm (c, d and h), 500 nm (f and g).
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dishes. The hepatic tissue sheets showed significantly greater hepatocyte-specific function in terms of protein expression and drug metabolism than did the conventionally harvested hepatocytes (Fig. 2).
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Figure 2 In vitro comparison of the function of isolated hepatocytes versus hepatic tissue sheets. (a,b) Functional assessment of cultured hepatocytes harvested as hepatic tissue sheets or single hepatocytes under a conventional method using collagenase. hA1AT mouse hepatocytes were cultured at a ratio of 8 105 hepatocytes per 35-mm-diameter PIPAAm culture dish. Hepatic tissue sheets were harvested by lowering the culture temperature to 20 1C for 15 min, whereas individual hepatocytes were collected by standard collagenase treatment before re-plating on Primaria culture dishes. The culture medium was harvested 24 h later, and hA1AT concentration (a) and albumin concentration (b) were measured. At the same time, the culture medium was replenished with the addition of 1 mg of lidocaine. (c) Residual lidocaine concentrations in the culture medium were measured 4 h later, and metabolized lidocaine doses were calculated (n ¼ 6–8 each). *P o 0.01 between groups.
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hepatocytes on the PIPAAm-grafted culture dishes. When the cultured hepatocytes reached confluency, we lowered the culture temperature to 20 1C for 15 min, which resulted in the spontaneous detachment of the hepatic tissue sheet from the plates (Fig. 1a). Histological examination showed that the harvested hepatic tissue sheet was composed of hepatocytes that had retained their cell-to-cell interactive connections (Fig. 1b). We confirmed maintenance of the hepatocyte-specific phenotype by positive staining for hA1AT and albumin (Fig. 1c,d). Fibronectin, which is important for cell adhesion, remained attached (Fig. 1e), unlike what occurs with normal cell detachment approaches using proteolytic enzymes such as collagenase that degrade the fibronectin during cell release from the plates. Ultrastructural examination showed the maintenance of hepatocyte microstructures, including microvilli and mitochondria, as well as intercellular functional connections including gap junctions, desmosomes and bile canaliculi (Fig. 1f). More importantly, extracellular matrix components that were deposited during hepatocyte culture were present beneath the tissue sheets (Fig. 1g). We confirmed the potential for clinical applicability of this technology, via the ability to engineer human hepatic tissue sheets, by culturing primary human hepatocytes from two individual donors on the PIPAAm-grafted surfaces (Fig. 1h). Finally, we also assessed the functionality of the hepatic tissue sheets, compared to the same number of hepatocytes harvested from the PIPAAm dishes by conventional collagenase methods, by re-plating them onto new culture
Scaffold-free approach for hepatic tissue engineering As indicated by our previous experiences, the establishment of a vascularized compartment within the subcutaneous space is an important step in the development of an ectopic liver system in that it enhances hepatocyte engraftment and allows for persistent viability after transplantation without loss of hepatocellular function5,6. We first developed the vascularized cavity within the subcutaneous space of wild-type FVB/N mice by implanting a basic fibroblast growth factor (bFGF)-releasing device5. Upon removal of the device, we transferred a freshly harvested monolayer sheet of hepatic tissue using a support membrane for insertion and transplantation into the vascularized cavity. The survivability of the transplanted tissue in vivo was found to persist for at least 235 d (Fig. 3a), as determined by the measurement of the recipient serum hA1AT, a marker specific for the donor hepatocytes5–9. Subsequently, we transplanted 8 105 freshly isolated hepatocytes (the same number of cells used to create each tissue sheet) into the liver through the portal vein and compared the serum hA1AT of recipients with that of mice transplanted with the hepatic tissue sheets. At day 28, the hepatic tissue sheet engineering approach resulted in significantly higher serum hA1AT than did conventional intrahepatic transplantation of freshly isolated hepatocytes (Fig. 3b). Histological examination at day 120 revealed that a two-dimensional tissue monolayer composed of healthy hepatocytes was engineered in the subcutaneous space (Fig. 3c). The detection of hA1AT and albumin in the engineered tissue showed that the hepatocyte phenotype was maintained (Fig. 3d,e). More importantly, robust
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h Figure 3 Morphological and functional characteristics of the engineered hepatic tissue sheets following transplantation into the subcutaneous space. (a) Maintenance of the functional volume of the engineered hepatic tissues as measured by mouse serum hA1AT levels after tissue transplantation. (J, ) Mice received a piece of a hepatic tissue sheet into the vascularized subcutaneous compartment in two different experiments (n ¼ 5 mice/ experiment); (n) mice received a piece of a hepatic tissue sheet (same experiment as J) into normal, nonvascularized subcutaneous space (n ¼ 5). (b) Engraftment of freshly isolated hepatocytes transplanted into the liver through the portal vein, or hepatic tissue sheets transplanted into the vascularized subcutaneous cavity. Hepatocytes (8 105) were transplanted in both groups and examined at day 28 (n ¼ 6 mice per group). (c–g) Histological analyses of the engineered hepatic tissue at day 120 from the mouse group (J in a) engrafted with the hepatic tissue sheet into the vascularized subcutaneous cavity. (c) Hematoxylin-eosin staining. (d) hA1AT immunostaining. (e) Albumin staining. (f) CYP2B (left) and CYP1A (right) immunohistochemical analyses of the engineered hepatic tissues in mice that received three consecutive days of intraperitoneal injections of either phenobarbital or 3-methylcholanthrene, respectively. (g,h) Immunofluorescent staining for BrdU (red) and hA1AT (green) of the engineered hepatic tissues in mice that received two-thirds hepatectomy at day 106 (g) or sham operation as a control at day 106 (h) followed by continuous infusion of BrdU for 2 weeks. *P o 0.01 versus the other two groups. #P o 0.05 between groups. Scale bars: 500 mm (c), 100 mm (d–h).
Figure 4 Stacking multiple monolayers of hepatic tissue for the engineering of three-dimensional hepatic tissues. (a) Engineered hepatic tissue volume determined by recipient serum hA1AT. Single (n) or double (J) hepatic tissue sheets were transplanted into the vascularized subcutaneous cavity. (b,c) Histological analysis of the engineered hepatic tissue at day 10 in mice engrafted with double layers of the hepatic tissue sheets within the vascularized subcutaneous cavity. Hematoxylin-eosin staining (b) and periodic acid–Schiff (PAS) staining (c). (d) Hematoxylin-eosin staining for histological analyses of the engineered hepatic tissue at day 140 in mice engrafted with double layers of the hepatic tissue sheets within the vascularized subcutaneous cavity (n ¼ 6 each). (e) Hepatic tissue engraftment after transplantation into the vascularized subcutaneous cavity. Freshly isolated hepatocytes suspended in Matrigel extracellular matrix components or four hepatic tissue sheets were stacked, transplanted into the vascularized cavity and examined at day 21. The same number of hepatocytes (3.2 106) were transplanted in both groups (n ¼ 7 in suspended hepatocyte group and n ¼ 4 in the 4 tissue sheet group). *P o 0.01 between groups at all time points throughout the experiment. #P o 0.005 between groups. Scale bars: 1 mm (b–d).
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Engineering three-dimensional hepatic tissues Subsequently, we investigated whether stacking multiple monolayers of the hepatic tissue would allow the liver system to be formed in three dimensions. We performed tissue sheet layering in the subcutaneous cavity in vivo by transferring the second hepatic tissue sheets onto the freshly transplanted initial tissue sheets. The multiplied hepatic tissue sheets remained viable and showed higher functional tissue volume as compared with the single monolayer hepatic tissues (Fig. 4a). We also confirmed the functionality of the three-dimensional hepatic tissues by a healthy morphology (Fig. 4b) and positive staining for glycogen (Fig. 4c). More importantly, the layered hepatic tissue sheets formed a large three-dimensional hepatic tissue mass and persisted stably for longer than 140 d (Fig. 4a,d). Additionally, we observed numerous blood vessels both within the engineered hepatic tissues and surrounding the threedimensional tissue masses. We did not detect the formation of a biliary structure within or surrounding the de novo–engineered tissue area.
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induction of CYP2B and CYP1A (Fig. 3f) was found following intraperitoneal inoculation with phenobarbital or 3-methylcholanthrene, respectively, into the recipient mice. These data demonstrated the ability of the ectopically engineered hepatic tissues to take up and metabolize circulating chemical compounds. Another important liver-specific feature found in the engineered two-dimensional hepatic tissue was its ability to respond to a regenerative signal. We induced a regenerative stimulus by performing a two-thirds liver resection in the recipient mice at day 106 and delivered bromodeoxyuridine (BrdU) for 14 d. In the engineered liver tissues, 52.4 ± 3.7% of the hepatocytes were labeled with BrdU after the twothirds liver resection (Fig. 3g). In contrast, in the sham-operation group, only 9.5 ± 3.2% of the cells had BrdU labeling (Fig. 3h). Furthermore, immunofluorescence staining for hA1AT showed that the engineered hepatic tissues formed stratified structures composed of several cell layers after the two-thirds hepatectomy. Taken together, these data clearly suggested that the engineered hepatic tissues had the ability to proliferate and grow in response to a regenerative event.
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TECHNICAL REPORTS Finally, we compared the efficacy of the present three-dimensional tissue engineering approach to previous methods using isolated hepatocytes suspended in Matrigel extracellular matrix components. In contrast to the hepatic tissue sheets, when we suspended less than 1.5 106 freshly isolated hepatocytes in Matrigel and implanted them into the vascularized cavity, the transplanted hepatocytes did not show persistent survival (data not shown). We therefore transplanted 3.2 106 freshly isolated hepatocytes suspended in Matrigel into the vascularized subcutaneous cavity created by the bFGF-releasing device, as reported before5. For comparison, we then engineered threedimensional hepatic tissues by stacking four hepatic tissue sheets (3.2 106 hepatocytes total). As indicated by significantly higher serum hA1AT at day 21, the layered hepatic tissue sheets showed superior functional volume over the freshly isolated hepatocytes suspended in Matrigel (Fig. 4e). DISCUSSION In tissue engineering, subcutaneous locations should be recognized as an important ectopic site to target because of the minimal amount of risk to the individual as well as the relative ease of cell implantation. In situations requiring multiple transplantations to support the impaired liver status of the individual19, it is also beneficial that this approach would also require only simple, noninvasive procedures. Here, we successfully re-created functional liver tissues in the subcutaneous space using bioengineered hepatic tissue sheets. Several advantages of our present approach, as compared with previous work in the area, can be noted. First, the protocol to engineer the tissue sheets was simple and could be performed without biodegradable scaffolds or extrinsic extracellular components. Second, the de novo formation of a subcutaneous vascular network, allowing for the recruitment of endothelial cells by the removable bFGF-releasing device made possible the long-term (4200 d) support and viability of the transplanted hepatic tissues. Third, the engineered hepatic tissues showed several characteristics of liver functionality, including the ability to take up circulating chemical compounds followed by drug metabolism–related enzyme expression, as well as mediating proliferation in response to regenerative stimuli. Finally, the stacking of hepatic tissue sheets allowed for the formation of a three-dimensional miniature liver system that maintained its biological function over a prolonged period of time. In our approach, the highly vascularized subcutaneous cavity had a great impact on the maintenance of the two- and three-dimensional hepatic tissues. We and others have recently shown in in vitro studies that various hepatocyte-specific functions could be maintained when isolated primary hepatocytes were cultured with endothelial cells18,20. It has been documented21 that growth factor interactions between the two types of cells and the creation of sinusoidal surfaces in hepatocytes mediate the functional maintenance. In this regard, multiplying hepatic sheets in close proximity to endothelial cells may help to strategically create integrated hepatic tissues. An important issue in hepatic tissue engineering remains the limitations on plasma exchange between the engineered tissues and the general circulation, because a high volume of blood circulation through the liver is normally required. In this context, extracorporeal hepatocyte-based bioartificial liver support systems have been developed to temporarily support individuals with acute liver failure. These bioartificial liver support systems provide an advantage over the present approach, because they support a high volume of plasma flow through the devices. The complex design of these large devices, as well as their limited periods of therapeutic efficacy (on the scale of days), however, have prompted us to develop this simple and basic approach.
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Because both the two- and three-dimensional functional hepatic tissues created here are relatively small in size, plasma exchange may be limited and therefore incompatible with some clinical applications. Nevertheless, the successful demonstration of CYP1A and CYP2B enzyme induction within the engineered liver tissues clearly shows the presence of a functional plasma exchange system that allows the hepatic tissues to take up circulating chemicals. We also clearly showed here that stacking multiple tissue sheets during transplantation procedures results in significantly higher functional hepatic tissue volume than is obtained with individual tissue sheet transplantation. The determination of whether these three-dimensional tissues created by multiple tissue sheets can develop structural polarity, such as sinusoidal and biliary surfaces, will, however, require further investigation. Presently, the engineering of tissue sheets cannot replace wholeorgan transplantation because it does not generate biliary connections to the intestine. It is clear from our results, however, that this method is a significant improvement over conventional methods of isolated hepatocyte transplantation. Moreover, our approach does not require the infusion of cells into the circulation, minimizing the risk of donor cell embolization in the portal circulation or the lungs, which can often accompany conventional hepatocyte transplantation procedures2,22,23. In the past decade, considerable progress has been made in bringing hepatocyte-based therapy to the bedside2,24. Clinical trials in which a small number of allogenic hepatocytes have been transplanted into the liver through the portal vein have clearly shown therapeutic benefit to individuals with congenital metabolic diseases, such as Crigler-Najjar syndrome22, glycogen storage disease25 and congenital deficiency of coagulation factor VII26. It has also been experimentally shown that engineering small hepatic tissues under the kidney capsule could provide therapeutic effects on hemophilia6. Because our approach involves the incubation of the primary isolated hepatocytes for several days on PIPAAm culture dishes, this would also provide the opportunity to genetically modify the isolated hepatocytes using either viral or nonviral gene therapy technologies, in cases where the hepatocytes are isolated from persons with inherited genetic liver diseases. Based on our previous work in genetically modifying isolated hepatocytes with gene therapy vectors27, these inherited liver diseases will benefit from the development of hepatic tissue engineering approaches using autologous hepatocytes. The functional hepatic tissue volume required to achieve therapeutic effects, however, will have to be optimized for each specific liver disease. Additionally, although therapeutic approaches using only autologous cells and free of biodegradable scaffolds theoretically reduces the risk of host inflammation and fibrosis, detailed investigations of the practical benefits are warranted. In conclusion, here we describe a new approach to create a uniformly continuous sheet of hepatic tissue in vitro, which could be transplanted into the subcutaneous space of mice to develop into a more spatial two- or three-dimensional miniature liver system. Although further studies in a large-animal model will be needed to confirm the potential therapeutic efficacy, this new approach to engineering miniature ectopic liver systems from individual isolated hepatocytes presents a concept that may possibly be applied as a new-generation therapy for liver diseases, particularly congenital metabolic diseases. METHODS Animals. We used transgenic mice expressing human a1-antitrypsin driven by the A1AT hepatocyte-specific promoter (hA1AT-FVB/N, H-2q) as hepatocyte donors28. We used wild-type female FVB/N mice (10–12 weeks), which have a background syngeneic to that of the hA1AT-FVB/N mice, as recipient animals.
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We conducted all mouse experiments in accordance with the institutional guidelines set forth by the Nara Medical University Animal Care Committee. Preparation of temperature-responsive cell culture surfaces. Procedures for the preparation of temperature-responsive polymer (PIPAAm)-grafted culture surfaces have been described15. Briefly, we spread N-isopropylacrylamide monomer (provided by Kohjin Co., Ltd.) in 2-propanol solution onto polystyrene cell culture dishes (35-mm diameter, Falcon 3001; Becton Dickinson). We then irradiated the dishes (0.3 MGy electron beam dose) using an Area Beam Electron Processing System (Nisshin–High Voltage), vigorously rinsed them with cold distilled water to remove ungrafted monomer, and sterilized them with ethylene oxide gas before experimental use. Hepatocyte isolation and creation of two-dimensional hepatic tissue sheets. We isolated primary mouse hepatocytes from donors as described5–9. We separated isolated hepatocytes from nonparenchymal cells by three rounds of low-speed centrifugation at 50g. We used hepatocytes with viabilities 490% as quantified by trypan blue dye exclusion test in all experiments. We cultured the hepatocytes on PIPAAm culture dishes at a ratio of 8 105 cells per dish at a temperature of 37 1C. When the plated hepatocytes reached confluency 72 hours later, we detached the cultured hepatocytes from the culture dish and harvested them as a uniformly connected tissue sheet by lowering the culture temperature to 20 1C for 15 min. We obtained human liver tissues from individuals after acquiring their written informed consent for experimental use of harvested liver samples. We isolated primary human hepatocytes from the harvested liver samples, as described8, and used human hepatocytes isolated from two individuals for the in vitro protocols to engineer the hepatic tissue sheets. After separating the hepatocytes from nonparenchymal cells, we cultured them on PIPAAm dishes using the procedures described above. Functional evaluation of the hepatic tissue sheets in vitro. We harvested the hepatic tissue sheets from PIPAAm culture dishes by low-temperature treatment and re-plated each individual sheet on separate Primaria culture dishes (Becton Dickinson). We also harvested hepatocytes from PIPAAm culture dishes by standard collagenase treatment (collagenase S-1; Sigma). We collected suspended cells by gentle pipetting, and followed this with immediate collagenase inactivation by the addition of serum-containing medium. After centrifugation at 50 g, we re-plated the hepatocytes harvested by collagenase treatment on Primaria culture dishes. We measured the secretion of albumin and hA1AT into the culture medium for 24 h. To determine lidocaine metabolism, we replenished the culture medium in some re-plated cultures with the addition of 1 mg of lidocaine (AstraZeneca). Four hours later, we measured residual lidocaine concentrations in the culture medium by fluorescence polarization immunosorbent assay as described29. bFGF-releasing device for vascular network induction. To create a vascularized cavity as a platform for tissue transplantation, we prepared a mesh device that provides the gradual release of bFGF5. In brief, we manufactured small bags from polyethylene terephthalate mesh (Nippon Tokushu Fabric) with three-dimensional configuration of 20 15 1 mm3 coated internally using 3% polyvinyl alcohol hydrogel (Yunitika). We dissolved 20 mg bFGF (Kaken Pharmaceutical) in 5% agarose (LT-600; Shimizu Shokuhin) and, immediately upon solubilization, we inserted the bFGF-agarose mixture into the mesh device and implanted it under the subcutaneous space of the recipient mice. Ten days later, we obtained a highly vascularized subcutaneous platform by removing the bFGF-releasing device. Hepatic tissue sheet transplantation procedures. We anesthetized wild-type female FVB/N mice that had received a bFGF-releasing device in the dorsal subcutaneous space. We exposed the highly vascularized subcutaneous platform by making an L-shaped skin incision (1.5 2 cm) to open the subcutaneous site, and then removed the device. Before the surgical procedures, we detached the mouse hepatic tissue sheets from PIPAAm culture dishes by incubation at 20 1C for 15 min. We attached the recovered tissue sheets to poly(vinylidene difluoride) support membranes (Millipore) and transplanted them onto the vascularized platform. Five minutes after transplantation, the hepatic tissue sheets formed stable attachment to the subcutaneous site, and we
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simply removed the overlying poly(vinylidene difluoride) membrane. We transplanted additional tissue sheets directly over the first using the same procedures to make bilayer hepatic tissues. In some experiments, we repeated the procedures to make four-layer hepatic tissues composed of 3.2 106 hepatocytes. Alternatively, we resuspended 3.2 106 freshly isolated hepatocytes in cold medium and cold Engelbreth-Holm-Swarm (EHS) gel (Matrigel; Becton Dickinson) and transplanted them into the vascularized subcutaneous space induced by the bFGF-releasing device. Detailed methods for evaluation of the transplanted hepatic tissue sheets and statistical analysis are described in Supplementary Methods online. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS The authors thank H. Sakai (CellSeed Inc.) for optimizing the PIPAAm culture dish conditions; G.L. Bumgardner (Ohio State University) for the hA1AT-FVB/N mouse line; and Y. Murakami (Kyoto University) for technical supervision in the development of the bFGF-releasing mesh device. This work was supported in part by Scientific Research Grants No. 15390632 (Y.N.) and No. 25691269 (K.O.), the Center of Excellence (COE) Program for the 21st Century (T.O.), and the Leading Project (K.O., M.Y., T.O.), from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and the Terumo Life Science Foundation (K.O.). AUTHOR CONTRIBUTIONS K.O., M.Y., T.O. and Y.N. designed the research; K.O., T.Y., H. Kuge, H. Kanehiro and Y.N. performed the hepatic tissue sheet experiments; M.Y., J.Y. and T.O. developed the temperature-responsive culture dishes; M.T. and T.A. performed the histological analyses; H.I. manufactured and provided the bFGF-releasing devices; K.O., T.Y., H. Kuge and J.Y. interpreted and analyzed the data; and K.O., M.Y. and J.Y. wrote and drafted the manuscript. COMPETING INTERESTS STATEMENT The authors declare competing financial interests: details accompany the full text HTML version of the paper at http://www.nature.com/naturemedicine. Published online at http://www.nature.com/naturemedicine/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions
1. Strom, S. & Fisher, R. Hepatocyte transplantation. New possibilities for therapy. Gastroenterology 124, 568–571 (2003). 2. Ohashi, K., Park, F. & Kay, M.A. Hepatocyte transplantation: clinical and experimental application. J. Mol. Med. 79, 617–630 (2001). 3. Griffith, L.G. & Naughton, G. Tissue engineering—current challenges and expanding opportunities. Science 295, 1009–1014 (2002). 4. Gouon-Evans, V. et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm. Nat. Biotechnol. 24, 1402–1411 (2006). 5. Yokoyama, T. et al. In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity. Am. J. Transplant. 6, 50–59 (2006). 6. Ohashi, K. et al. Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases. Hepatology 41, 132–140 (2005). 7. Ohashi, K. et al. Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice. Cell Transplant. 14, 621–627 (2005). 8. Ohashi, K. et al. Sustained survival of human hepatocytes in mice: a model for in vivo infection with human hepatitis B and hepatitis delta viruses. Nat. Med. 6, 327–331 (2000). 9. Kuge, H. et al. Genetic modification of hepatocytes towards hepatocyte transplantation and liver tissue engineering. Cell Transplant. 15, 1–12 (2006). 10. Lee, H. et al. Effect of implantation site on hepatocytes heterotopically transplanted on biodegradable polymer scaffolds. Tissue Eng. 9, 1227–1232 (2003). 11. Yang, J. et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 26, 6415–6422 (2005). 12. Yamada, N. et al. Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol. Chem. Rapid Commun. 11, 571–576 (1990). 13. Okano, T., Yamada, N., Sakai, H. & Sakurai, Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J. Biomed. Mater. Res. 27, 1243–1251 (1993). 14. Nishida, K. et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N. Engl. J. Med. 351, 1187–1196 (2004). 15. Hirose, M., Kwon, O.H., Yamato, M., Kikuchi, A. & Okano, T. Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface. Biomacromolecules 1, 377–381 (2000).
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TECHNICAL REPORTS 16. Shimizu, T. et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ. Res. 90, e40–e48 (2002). 17. Miyahara, Y. et al. Monolayered mesenchymal stems cells repair scarred myocardium after myocardial infarction. Nat. Med. 12, 459–465 (2006). 18. Harimoto, M. et al. Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes. J. Biomed. Mater. Res. 62, 464–470 (2002). 19. Stephenne, X. et al. Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency. Gastroenterology 130, 1317–1323 (2006). 20. Nahmias, Y., Casali, M., Barbe, L., Berthiaume, F. & Yarmush, M.L. Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes. Hepatology 43, 257–265 (2006). 21. Michalopoulos, G.K. & DeFrances, M.C. Liver regeneration. Science 276, 60–66 (1997).
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