Three-dimensional reconstruction of a biliary system in a bioengineered liver using decellularized scaffold

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Liver scaffolds engineered through decellularization techniques have been developed for clinical applications; however, reconstruction of a functional biliary system remains a challenge. This study aimed to establish a three-dimensional biliary system in bioengineered livers through recellularization of rat primary hepatocytes (PHs) and liver ductal organoids (LDOs). Decellularized rat liver scaffolds were recellularized using rat PHs and green fluorescent protein (GFP)-expressing LDOs. Dissociated LDOs were injected via the bile duct and cultured for 5 days using a perfusion device, followed by PH injection and 2 days of culture. During co-culture, biliary drainage fluid and culture medium were collected to compare total bile acid concentrations using enzyme-linked immunosorbent assay. Histological and immunofluorescence analyses were performed after 7 days of perfusion culture. Histological analyses confirmed the engraftment of GFP-expressing LDOs into bile ducts and PHs into the parenchymal space. Engrafted PHs expressed ZO-1 and MRP2, forming bile canaliculi. In specific regions, MRP2-positive PHs and KRT19-positive LDO-repopulated cells adhered to each other, resembling the native liver structure. In the samples exhibiting such structures, total bile acid concentrations in the biliary drainage fluid tended to be higher than in the culture medium.This study suggested that a three-dimensional functional biliary system could be reconstructed in bioengineered livers. These findings represent a significant step toward the realization of bioengineered livers using decellularization and recellularization techniques. Biological sciences/Biological techniques Biological sciences/Biotechnology Health sciences/Gastroenterology Health sciences/Medical research Biological sciences/Stem cells Liver regeneration tissue engineering liver ductal organoids hepatocytes bioartificial organs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION End-stage liver disease (ESLD) causes approximately 2 million deaths annually worldwide [ 1 ]. Liver transplantation is the only curative and well-established treatment for patients with ESLD; however, in the United States alone, approximately 2,500 patients with ESLD die annually because of the shortage of donor organs [ 2 ]. Various approaches, including cell transplantation, bioartificial organs, and liver support devices, have been investigated to address this challenge [ 3 ]; however, none have been successfully established as therapeutic alternatives. Decellularized liver scaffold-based bioengineering has recently emerged as a promising strategy to address the needs of these patients [ 4 – 6 ]. These scaffolds, composed of extracellular matrix, retains its microvasculature and biological properties, providing a three-dimensional (3D) framework for recellularization with liver cells, such as hepatocytes, cholangiocytes, and endothelial cells [ 7 – 16 ]. In the native liver, coordinated interactions among these cells are crucial for maintaining functional complexity. Bile excretion is facilitated by the connection between the bile canaliculi, which are formed by hepatocytes, and the bile ducts (BDs), which are composed of cholangiocytes. Recent studies have reported that a bile excretion system could be constructed by connecting hepatocytes and cholangiocytes in a two-dimensional culture environment using different cell sources [ 17 , 18 ]. However, a 3D reconstruction of a biliary excretion system has not yet been achieved. We recently reported that mouse and human liver ductal organoids (LDOs) could be potential BD cell sources for recellularization [ 11 ]. LDOs, isolated from BD fragments embedded in Matrigel, could be cultured and passaged to maintain cholangiocyte characteristics. Recellularization with LDOs, following dissociation into single cells via the BD of the liver scaffold, can reconstruct the luminal structure of the BDs. Primary hepatocytes (PHs) are isolated cells that perform essential liver functions, including bile secretion, protein synthesis, and detoxification. Because of their ability to closely mimic the physiological and metabolic activities of the liver tissue, they are widely used for in vitro studies of liver function, drug metabolism, and toxicity. They have also been investigated for recellularization of cell sources. Previous studies have reported that transplantation of recellularized livers with PHs could support liver function in recipients [ 19 , 20 ]. However, the structural and functional integration of hepatocytes within recellularized livers, particularly regarding how these hepatocytes engraft, repolarize, and contribute to bile canaliculi formation, have not been thoroughly investigated. In this study, we hypothesized that co-recellularization of decellularized liver scaffolds with PHs and LDOs could reconstruct a 3D biliary system in bioengineered livers. RESULTS Decellularization of the rat whole liver A translucent acellular liver scaffold was generated 48 h after decellularization (Fig. 1 A). Hematoxylin eosin and Masson’s trichrome staining confirmed the absence of nuclei and cytoplasmic components in the decellularized liver scaffold (Fig. 1 B). Crystal violet injection of the portal vein (PV) and BD showed that the vascular system remained intact after decellularization (Fig. 1 C). Characteristics of rat LDOs Rat bile duct fragments were isolated after enzymatic digestion, as shown in Fig. 2 A and LDOs proliferated rapidly when cultured in expansion medium (EM) embedded in Matrigel (Fig. 2 B, Supplementary Table S1 ). LDOs in expansion culture were analyzed using immunofluorescence to determine the expression of markers specific to cholangiocytes and hepatocytes. Compared to native liver (Fig. 2 C), Immunofluorescence analyses revealed that the LDOs expressed cholangiocyte lineage markers KRT19 and SOX9, while the hepatocyte markers ALB and Hnf4a were absent (Fig. 2 D). These findings indicate that LDOs cultured under expansion conditions exhibited cholangiocytic characteristics. Bile canalicular formation in recellularized liver with PHs Freshly isolated rat PHs (5 × 10 7 cells) were injected via the BD of the scaffolds, followed by perfusion culture via the PV for 2 days (Fig. 3 A). Histological analysis demonstrated appropriate distribution of PHs within the parenchymal space, with some cells engrafted and adherent to each other (Fig. 3 B). Immunofluorescence revealed MRP2 and ZO-1 expression on the membrane side of viable hepatocytes, showing patterns similar to those of native liver (Fig. 3 C). Electron microscopy confirmed bile canalicular structures between hepatocytes, characterized as a space with microvilli surrounded by tight junctions, similar to the native liver (Fig. 3 D). Reconstruction of biliary structure in recellularized liver with PHs and LDOs To reconstruct biliary structure, 5 × 10 6 LDO-derived cells were injected via the BD and perfused with EM via the PV for 5 days. Then, freshly isolated rat PHs (5 × 10 7 cells) were injected via the BD, followed by perfusion culture with co-culture medium via the PV for 2 days (Fig. 4 A). Histological analyses revealed the distribution of cells engrafted into the BD lumen and parenchymal space. Immunofluorescence confirmed that GFP-positive cells corresponded to KRT19-positive cells and were distinct from albumin-positive cells (Figs. 4 B and 4 C). These results indicate that LDOs reconstructed the BDs and PHs were engrafted in the parenchyma, maintaining their respective characteristics. In some cell clusters, the cells engrafted into the BD and parenchyma adhered to each other. In these areas, MRP2 expression in the recellularized PHs was co-localized with KRT19-positive BDs, resembling native liver architecture (Fig. 4 D). Excretion of bile acids into recellularized BDs During the 2-day perfusion culture after recellularization of PHs and LDOs, approximately 30–50µL of drainage fluid from the BD was collected using a syringe connected to the culture dish (Fig. 5 A). ELISA for the total bile acids in the biliary drainage and culture medium were performed. In the three samples that exhibited bile canaliculi with MRP2 expression close to the BDs (Fig. 4 , the total bile acid concentrations in the biliary drainage tended to be higher than in the culture medium (6.35 ± 2.43 vs 2.59 ± 0.29nmol/10 6 cells, p = 0.100; Fig. 5 B). In contrast, among the five specimens lacking bile canaliculi in proximity to bile ducts, no significant difference in concentration was observed (3.87 ± 1.80 vs 5.73 ± 2.93nmol/10 6 cells, p = 0.310) (Supplementary Figure S1 ). DISCUSSION In this study, rat PHs injected into decellularized liver scaffolds adhered to each other, repolarized, and formed bile canaliculi. Co-recellularization with rat PHs and LDOs enabled the reconstruction of biliary structures, with BDs repopulated by LDOs and bile canaliculi formed by PHs within certain cell clusters. In samples where the biliary structure was successfully reconstructed, the total bile acid concentration in the biliary drainage from the recellularized liver tended to be higher than that in the perfusing culture medium. These findings suggest that a 3D biliary excretion system can be reconstructed in a bioengineered liver using decellularized scaffolds recellularized with rat PHs and LDOs. Over the past decade, significant advancements have been made in the development of decellularized liver-based bioengineered livers using various recellularized cell sources for clinical applications. Takeishi et al. reported that hepatocytes, cholangiocytes, and vascular endothelial cells derived from human induced pluripotent stem cells successfully recellularized rat liver scaffolds and could be transplanted into immunocompromised rats [ 12 ]. Human-specific albumin and A1AT were identified in the serum using ELISA 4 days after transplantation, indicating graft functionality in vivo. In a more clinically relevant study, Higashi et al. reported heterotopic transplantation of porcine recellularized liver grafts with PHs and vascular endothelial cells into porcine liver failure models [ 19 ]. Angiography and contrast-enhanced CT on postoperative day 28 confirmed graft perfusion, and serum markers such as total bilirubin, AST, ALT, ALP, and NH₃ were lower in the transplant group than in the control group at most postoperative time points. Despite these developments towards the adoption of bioengineered livers in clinical settings, the biliary system has not been sufficiently addressed. In this study, we first focused on how the recellularized hepatocytes engrafted onto the scaffold. We demonstrated that rat PHs can repolarize and form bile canaliculi, as shown by ZO-1 and MRP2 expression and transmission electron microscopy at the parenchymal space. These results indicate that the extracellular matrix of the liver scaffold facilitates hepatocyte engraftment and contributes to the formation of bile canaliculi. To the best of our knowledge, this is the first study to demonstrate the reconstruction of bile canalicular structures and their distribution within a recellularized liver. Two-dimensional reconstruction of a bile excretion system has been reported. Tanimizu et al. co-cultured mouse small hepatocytes and primary cholangiocytes in a two-dimensional culture environment, enabling the development of cell populations, which retained the functional connections of hepatocytes and cholangiocytes [ 17 ]. In their study, bilirubin and fluorescein-labeled bile acid were absorbed by hepatocytes, excreted into bile canaliculi, and accumulated in the biliary system. Huang et al. used chemically induced liver progenitors to engineer an integrated tubule-hepatocyte tissue [ 18 ]. Chemically induced liver progenitors were induced from mature hepatocytes, differentiated into biliary epithelial cells, and formed biliary duct-like structures. Rat PHs were then plated onto tubular biliary-duct-like structures and cultured for 5 days in two-dimensional culture conditions, resulting in the formation of integrated tubule-hepatocyte tissue with functional interaction between hepatocytes and chemically induced liver progenitor-derived biliary-duct-like structures. Three-dimensional reconstruction of a bile excretion system in bioengineered livers could significantly contribute not only to the adoption of bioengineered livers in transplantation medicine but also to the field of drug development. Several studies have attempted 3D reconstruction of the biliary system in decellularized liver scaffolds using recellularization techniques [ 21 – 23 ]; however, they did not demonstrate detailed biliary structure, bile canaliculi, and BD, or data suggesting functional reconstruction of the biliary excretion system. In this study, we used LDOs as recellularization sources for decellularized BDs. We previously reported that LDOs have cholangiocyte characteristics and can efficiently repopulate BDs of the liver scaffold [ 11 ]. An advantage of using LDOs is their genetic stability during long-term expansion, which allows a large cell supply. Another advantage is the potential to overcome immunogenicity, which is a major challenge in transplantation medicine, as LDOs can be isolated from human specimens for BD recellularization [ 11 , 24 ]. We demonstrated that serial recellularization of rat LDOs and PHs successfully generated tissues wherein the BDs repopulated with LDOs were in close proximity to bile canaliculi formed by PHs. A schematic diagram of the reconstructed bile excretion system in the recellularized liver is provided in Supplementary Figure S2. Hepatocytes spread into the parenchymal space, while reconstructed BDs connected with hepatocytes in specific areas. The arrangement of bile canaliculi and BDs closely resembled the native liver, suggesting the reconstruction of the biliary system. There have been no reports on the reconstruction of bioengineered livers with a functional bile excretion system. In vivo, bile is produced by hepatocytes, secreted into bile canaliculi, transported through BDs, and discharged into the intestinal tract. Bile acid concentration is higher in bile than in the bloodstream; thus, if the bile excretion pathway is successfully reconstructed, the concentration of bile acids in BD effluents should be higher than in the circulating medium. Our experiments demonstrated that only specimens where bile canaliculi formed in proximity to BDs showed a tendency toward higher bile acid concentrations in the biliary drainage. To our knowledge, this is the first study to evaluate bile excretion function in bioengineered livers generated from decellularized scaffolds. A limitation of this study is that real-time bile flow in recellularized livers was not assessed; therefore, future studies should address this using fluorescent bile acid excretion assays and by performing ex vivo and/or in vivo blood perfusion experiments. Additionally, the present study does not establish a method to efficiently generate samples in which bile canaliculi and bile ducts are located in close proximity, indicating that further refinement of the approach is required. Ideally, if the bile excretion system in recellularized livers can be successfully reconstructed using autologous hepatocyte sources, it would significantly enhance the clinical potential of bioengineered livers. In summary, our findings indicate the potential for reconstructing a 3D biliary structure in bioengineered livers generated through recellularization of liver scaffolds with rat PHs and LDOs. Bile excretion function was also suggested, representing a significant advance toward the realization of bioengineered livers. METHODS Animals Lewis rats (200–300 g; SLC, Hamamatsu, Japan) were used to prepare the 3D liver scaffolds, PHs, and LDOs. Rats were housed under controlled temperature and humidity conditions with a 12-hour light/dark cycle and were provided standard chow and water ad libitum at the Kyoto University animal facility. Rats used for liver procurement were euthanized by exsanguination under deep isoflurane anesthesia, in accordance with institutional guidelines and approved protocols. All animal experiments were approved by the Kyoto University Animal Experimentation Committee under protocols MedKyo22166 (valid from April 7, 2017 to March 31, 2023) and 24184 (from April 1, 2023 to March 31, 2025), and were performed in accordance with the Kyoto University Animal Protection Guidelines. The study is reported in accordance with ARRIVE guidelines. Liver procurement and decellularization Liver procurement and decellularization were conducted as previously reported [ 25 ]. After cannulation of the bile duct with a 24-gauge catheter and the portal vein with a 20-gauge catheter, livers were flushed with PBS. The organs were frozen at − 80°C, thawed overnight at 4°C, and then subjected to enzymatic perfusion with 0.25% (w/v) trypsin–1 mmol/L EDTA–4Na containing phenol red (Wako, Osaka, Japan) at 37°C for 1 h. Detergent perfusion was subsequently performed using 0.1% polyoxyethylene octyl phenyl ether (Wako) and 0.05% EDTA (Sigma, St. Louis, USA) at 0.5 mL/min for 48 h. Decellularized grafts were sterilized by perfusion with 0.1% peracetic acid (Sigma-Aldrich) for 2 h, followed by extensive PBS washing. Preparation of cell sources PHs Rat PHs were isolated using a two-step collagenase perfusion technique, as previously described [ 26 ]. Lewis rats were anesthetized with isoflurane (Wako), and the portal vein was cannulated with a 25-gauge needle. After transection of the suprahepatic and infrahepatic inferior vena cava, the liver was perfused with 50 mL of Ca²⁺-free HBSS containing 0.5 mM EGTA (Wako) and 2 U/mL heparin (Mochida, Tokyo, Japan) for 5 min, followed by 50 mL of a collagenase-based digestion buffer at 37°C for 10 min. The digestion buffer consisted of 0.15% dispase II (Sanko Junyaku, Tokyo, Japan), 0.15% collagenase type II (Gibco, Palo Alto, CA, USA), 0.1 mg/mL DNase I (Sigma, St. Louis, USA), 2 U/mL heparin, and physiological salts and glucose (NaCl 150 mmol/L, KCl 5.4 mmol/L, NaHPO₄ 0.34 mmol/L, MgSO₄ 0.1 mmol/L, CaCl₂ 5.0 mmol/L, NaHCO₃ 4.2 mmol/L, glucose 5.6 mmol/L, and HEPES 10 mmol/L; Wako). After perfusion, the liver was excised, gently agitated to release cells, and passed through a 100 µm mesh filter (BD Falcon, Franklin Lakes, NJ, USA). The cell suspension was washed three times by centrifugation at 50×g for 3 min at 4°C. PHs were used immediately for recellularization. Green fluorescent protein (GFP)-expressing LDOs Rat liver ductal organoids (LDOs) were established based on previously described protocols in mice [ 11 ], with modifications. The left lateral liver lobe from Lewis rats was excised, cut into 2–3 mm pieces, and further fragmented by sequential passage through 18-, 20-, 22-, and 23-gauge needles. Tissue fragments were then enzymatically digested in DMEM (Wako, Osaka, Japan) containing 2.6 µU/mL Liberase DH (Roche, Basel, Switzerland) and 0.1 mg/mL DNase I (Sigma-Aldrich, St. Louis, MO) at 37°C for 10 min. The resulting suspension was filtered through 100 µm and 40 µm meshes, and collected clusters were pelleted by centrifugation at 440 × g for 3 min. Approximately 20–30 clusters were resuspended in 7 µL of Matrigel-GFR (Corning, NY) and seeded on non-treated culture plates (IWAKI, Shizuoka, Japan). After polymerization at 37°C for 10 min, embedded clusters were cultured in expansion medium (EM) for 5–7 days until organoid formation. Organoids were passaged every 4–6 days using TrypLE Express (Invitrogen, Carlsbad, CA, USA) at 37°C for 15 min, and re-embedded in Matrigel-GFR for further expansion or recellularization. For genetic labeling, a GFP-expressing lentiviral vector (pLX304-GFP) was constructed by transferring the GFP coding sequence from pALB-GFP (#55759; Addgene) to pENTR4 (#17424; Addgene), followed by Gateway cloning into pLX304 (#25890; Addgene). Lentiviral particles were produced in HEK293FT cells co-transfected with pLX304-GFP, psPAX2 (#12260; Addgene), and pMD2.G (#12259; Addgene) using X-tremeGENE™ HD (Roche), according to the manufacturer’s instructions. Viral supernatants were collected at 48 and 72 h after transfection and filtered through 0.45 µm PVDF membranes (Merck Millipore). For transduction, LDOs were dissociated into single cells and incubated with the viral supernatant supplemented with 10 µM Y-27632 (LC Laboratories, Woburn, MA), 1 mM N-acetyl-L-cysteine (Wako), and 10 µg/mL polybrene (Sigma). The cell–virus mixture was transferred to untreated plates and centrifuged at 2000 rpm for 1 h at room temperature. After centrifugation, the supernatant was removed, and the cells were embedded in Matrigel-GFR and cultured in EM. Organoids were subsequently expanded and selected with blasticidin (Wako). Recellularization and perfusion culture of rat whole liver Perfusion culture of recellularized liver with only PHs Recellularization was performed based on our previously reported method [ 11 ], with modifications. A total of 5.0 × 10⁷ primary hepatocytes were resuspended in 30 mL of HCM™ medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and infused into the liver scaffold via the bile duct at a rate of 1 mL/min. Following cell injection, the graft was maintained under static conditions at 37°C for 3 h to allow cell attachment. The recellularized liver was then connected to a perfusion circuit through the portal vein cannula and continuously perfused with HCM TM -based medium containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin using a peristaltic pump (Perista pump, ATTA, Japan) at 0.7–1.0 mL/min at 37°C. The perfusate exiting through the inferior vena cava was collected and recirculated within the system. Perfusion culture was maintained for 48 h. Perfusion culture of recellularized liver with LDOs and PHs Single-cell suspensions of LDOs (3–5 × 10⁶ cells) were prepared in 5 mL of EM and infused into the liver scaffold through the BD at a flow rate of 1 mL/min. The graft was then maintained under static conditions in EM for 3 h before being connected to the perfusion circuit as described above. The culture medium was replaced every other day, and perfusion culture was continued for 5 days. Subsequently, 5.0 × 10⁷ rat primary hepatocytes resuspended in 30 mL of co-culture medium (Supplementary Table S2) were infused via the BD. After static incubation for 3 h, perfusion culture was resumed with co-culture medium and maintained for an additional 48 h. Collection of drainage fluids from the BD During perfusion culture, the BD of the scaffolds recellularized with PH and LDO was connected to a syringe via a culture dish, and negative pressure was applied by pulling the plunger of the 1 mL syringe and securing it with a thread. Histological analysis Organoids were isolated from Matrigel, re-embedded in Cellmatrix Type I-A (Nitta Gelatin), and fixed overnight in 10% formalin (Wako) at room temperature. Recellularized liver grafts were fixed in 4% paraformaldehyde (Wako) for 24 h at 4°C and subsequently embedded in paraffin. Paraffin blocks were sectioned at 4 µm, deparaffinized, rehydrated, and subjected to hematoxylin and eosin staining or immunohistochemistry. For immunostaining, antigen retrieval was performed by autoclaving at 121°C for 15 min. Sections were blocked with PBS containing 10% donkey or goat serum and 0.1% Triton X-100 (Nacalai Tesque, Kyoto, Japan), followed by overnight incubation at 4°C with primary antibodies diluted in PBS supplemented with 5% donkey or goat serum and 0.1% Triton X-100. Secondary antibodies were applied for 1 h at room temperature. Nuclei were counterstained and mounted with ProLong™ Gold Antifade Mountant containing DAPI (Invitrogen), and images were acquired using an Olympus BX50F4 microscope (Olympus, Tokyo, Japan). For each sample, five sections representing all hepatic lobes were analyzed to ensure unbiased histological evaluation. The list of antibodies used is provided in Supplementary Table S3. Electron microscopy Electron microscopy was carried out as previously described [ 8 ]. Fresh and decellularized livers from Lewis rats were fixed overnight at 4°C in PBS containing 2% glutaraldehyde (Nacalai Tesque) and 4% paraformaldehyde. Fixed tissues were microdissected, dehydrated through a graded ethanol series (50, 60, 70, 80, 90, 99, and 100%), and immersed in t-butanol. Samples were then frozen at − 20°C, followed by sublimation. After sputter coating with a platinum–palladium alloy using an ion coater (JBC-3000FC, JEOL, Japan), specimens were examined with a scanning electron microscope (JSM-7900F, JEOL). Enzyme-linked immunosorbent assay (ELISA) of total bile acids After 48 h of perfusion culture of scaffolds recellularized with PHs and LDOs, culture medium and BD drainage fluids were collected. Total bile acid concentrations in both samples were measured using a Total Bile Acids Assay Kit (Cell Biolabs, San Diego, CA, USA) via ELISA. Background bile acid concentrations derived from FBS were measured in medium alone and subtracted from the values obtained for all samples. Bile acid concentrations were normalized to the number of engrafted hepatocytes (per 10 6 cells). Statistical methods Data are expressed as mean ± standard error of the mean. Statistical analyses were performed using R version 4.1.0 (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was defined as p < 0.05. Total bile acid concentrations were compared using an unpaired t -test. Abbreviations 3D, three-dimensional; BD, bile duct; ELISA, enzyme-linked immunosorbent assay; EM, expansion medium; ESLD, end-stage liver disease; GFP, green fluorescent protein; LDO, liver ductal organoids; PH, primary hepatocytes; PV, portal vein. Declarations ACKNOWLEDGMENTS We thank the Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University, for technical assistance with the analysis of electron microscopy data. FUNDING This work was supported by JSPS KAKENHI [grant numbers 22K08689, 24KJ1416]. Author contributions Conceptualization: Ken Fukumitsu Data curation: Hiroshi Horie, Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue Funding acquisition : Hiroshi Horie, Ken Fukumitsu Investigation : Hiroshi Horie, Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hidenobu Kojima, Satoshi Ogiso, Elena Yukie Uebayashi, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano Methodology : Hiroshi Horie, Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hidenobu Kojima, Satoshi Ogiso, Elena Yukie Uebayashi, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano Project administration : Ken Fukumitsu Resources: Hiroshi Horie, Ken Fukumitsu, Masahiro Inoue Supervision : Masahiro Inoue, Takamichi Ishii, Etsuro Hatano Writing - original graft: Hiroshi Horie, Ken Fukumitsu Writing - review and editing: Satoshi Ogiso, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano Competing interests The author declares no competing interests Data availability statement The data that support the findings of this study are available from the corresponding author, K.F., upon reasonable request. 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Supplementary Files Supplement.docx Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Dec, 2025 Reviews received at journal 08 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviews received at journal 30 Nov, 2025 Reviews received at journal 21 Nov, 2025 Reviewers agreed at journal 16 Nov, 2025 Reviewers agreed at journal 13 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviewers invited by journal 10 Nov, 2025 Editor assigned by journal 10 Nov, 2025 Editor invited by journal 07 Oct, 2025 Submission checks completed at journal 01 Oct, 2025 First submitted to journal 01 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7659575","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":545978498,"identity":"e8b77705-3cad-4d94-8518-e61b3c418c40","order_by":0,"name":"Hiroshi Horie","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Horie","suffix":""},{"id":545978499,"identity":"de39614c-b8d1-448c-acc6-8f72bfabb30a","order_by":1,"name":"Ken 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1","display":"","copyAsset":false,"role":"figure","size":1783501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of the decellularized liver scaffold.\u003c/strong\u003e (A) Gross appearance of the rat whole liver during decellularization. (B) Hematoxylin and eosin staining and Masson’s trichrome staining. (C) Intrahepatic portal tree and BDs stained with crystal violet. Scale bars = 50 μm. PV, portal vein; BD, bile duct.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7659575/v1/988b2c72c9a2d5ae72cb9e0f.png"},{"id":96366020,"identity":"b1645ea7-4227-4755-9825-ca4c8ed75e11","added_by":"auto","created_at":"2025-11-20 10:11:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5227694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation, culture, and characteristics of rat liver ductal organoids. \u003c/strong\u003e(A) Fragments of intrahepatic bile ducts precipitated in a conical tube. (B) Images at day 4 (left) and day 7 (middle). After three passages (right), sufficient purification was achieved. (C) Immunofluorescence showing hepatocytes positive for albumin and HNF4α, and cholangiocytes positive for KRT19 and SOX9 in the native liver. (D) Rat liver ductal organoids exhibited cholangiocyte characteristics (KRT19 and SOX9 expression) without hepatocyte characteristics (albumin and HNF4α expression). Scale bars = 50 μm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7659575/v1/8236455f48d217555735253a.png"},{"id":96366296,"identity":"736d1b13-a14f-4e79-8d53-a2b2ac7ff895","added_by":"auto","created_at":"2025-11-20 10:11:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4844431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecellularization with rat primary hepatocytes.\u003c/strong\u003e (A) (A-a) Cannulated rat liver before the two-step collagenase perfusion for primary hepatocyte preparation. (A-b) Extirpated rat liver after the perfusion. (A-c) Injection of isolated hepatocytes into the bile ducts of the decellularized liver at a constant rate using a syringe pump. (A-d) Recellularized liver in a customized chamber connected to a circulation device. (B) Gross appearance of recellularized liver. (C) Hematoxylin and eosin staining of recellularized liver. (D) Immunofluorescence of ZO-1 and MRP2 in native liver and recellularized liver. (E) Transmission electron microscopy images of the bile canalicular space with microvilli in native and recellularized livers. Scale bars = 50 μm (B-D) and 1 μm (E).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7659575/v1/fb3c7f55a8842e87eef67522.png"},{"id":96367138,"identity":"799b3b3c-8a5e-4cd0-bf6b-8c3969bfebba","added_by":"auto","created_at":"2025-11-20 10:12:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4510960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecelluralization with rat primary hepatocytes and liver ductal organoids. \u003c/strong\u003e(A) Gross appearance of the recellularized liver scaffold with hepatocytes and liver ductal organoids. (B and C) Immunofluorescence staining of (A) with indicated antibodies. (D) Hematoxylin and eosin staining of a region showing attachment of engrafted PH and LDO-derived bile duct, as indicated by the red circle. Bile duct cells and primary hepatocytes are indicated by arrows and arrowheads, respectively. (E) The expression patterns of MRP2 and KRT19 positive cells of native liver (left) and recellularized liver (right). Scale bars = 50 μm. GFP, green fluorescent protein, LDO, liver ductal organoid, PH, primary hepatocyte.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7659575/v1/2f4e6f51345fcfc4e13743c4.png"},{"id":96355242,"identity":"e38426e6-0873-4072-a44c-1fc641d5b551","added_by":"auto","created_at":"2025-11-20 08:16:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1618017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of total bile acid concentrations between bile duct discharge and culture medium, with histological correlation.\u003c/strong\u003e (A) Schematic illustration (left) and the actual image (right) showing a system for collecting bile duct discharge. (B) Comparison of total bile acids in the culture medium and bile duct discharge. Samples were collected from the cases in which MRP2-positive bile canaliculi were confirmed to be present adjacent to the KRT19-positive bile ducts (n = 3, \u003cem\u003ep \u003c/em\u003e= 0.100). Scale bars = 50 μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7659575/v1/8f5c86ae687057444a92aecf.png"},{"id":102785125,"identity":"ea80614c-a514-46d2-becb-e4799efc87eb","added_by":"auto","created_at":"2026-02-16 16:00:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19081958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7659575/v1/6c439db0-af17-4c96-b918-1e91029f2f93.pdf"},{"id":96355264,"identity":"3af9845f-574c-498f-ad40-0d12df28540c","added_by":"auto","created_at":"2025-11-20 08:16:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11851543,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-7659575/v1/895a0bc7a84d7c5cb8318070.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Three-dimensional reconstruction of a biliary system in a bioengineered liver using decellularized scaffold","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eEnd-stage liver disease (ESLD) causes approximately 2\u0026nbsp;million deaths annually worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Liver transplantation is the only curative and well-established treatment for patients with ESLD; however, in the United States alone, approximately 2,500 patients with ESLD die annually because of the shortage of donor organs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Various approaches, including cell transplantation, bioartificial organs, and liver support devices, have been investigated to address this challenge [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]; however, none have been successfully established as therapeutic alternatives.\u003c/p\u003e\u003cp\u003eDecellularized liver scaffold-based bioengineering has recently emerged as a promising strategy to address the needs of these patients [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These scaffolds, composed of extracellular matrix, retains its microvasculature and biological properties, providing a three-dimensional (3D) framework for recellularization with liver cells, such as hepatocytes, cholangiocytes, and endothelial cells [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the native liver, coordinated interactions among these cells are crucial for maintaining functional complexity. Bile excretion is facilitated by the connection between the bile canaliculi, which are formed by hepatocytes, and the bile ducts (BDs), which are composed of cholangiocytes. Recent studies have reported that a bile excretion system could be constructed by connecting hepatocytes and cholangiocytes in a two-dimensional culture environment using different cell sources [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, a 3D reconstruction of a biliary excretion system has not yet been achieved.\u003c/p\u003e\u003cp\u003eWe recently reported that mouse and human liver ductal organoids (LDOs) could be potential BD cell sources for recellularization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. LDOs, isolated from BD fragments embedded in Matrigel, could be cultured and passaged to maintain cholangiocyte characteristics. Recellularization with LDOs, following dissociation into single cells via the BD of the liver scaffold, can reconstruct the luminal structure of the BDs.\u003c/p\u003e\u003cp\u003ePrimary hepatocytes (PHs) are isolated cells that perform essential liver functions, including bile secretion, protein synthesis, and detoxification. Because of their ability to closely mimic the physiological and metabolic activities of the liver tissue, they are widely used for in vitro studies of liver function, drug metabolism, and toxicity. They have also been investigated for recellularization of cell sources. Previous studies have reported that transplantation of recellularized livers with PHs could support liver function in recipients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the structural and functional integration of hepatocytes within recellularized livers, particularly regarding how these hepatocytes engraft, repolarize, and contribute to bile canaliculi formation, have not been thoroughly investigated.\u003c/p\u003e\u003cp\u003eIn this study, we hypothesized that co-recellularization of decellularized liver scaffolds with PHs and LDOs could reconstruct a 3D biliary system in bioengineered livers.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDecellularization of the rat whole liver\u003c/h2\u003e\u003cp\u003eA translucent acellular liver scaffold was generated 48 h after decellularization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Hematoxylin eosin and Masson\u0026rsquo;s trichrome staining confirmed the absence of nuclei and cytoplasmic components in the decellularized liver scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Crystal violet injection of the portal vein (PV) and BD showed that the vascular system remained intact after decellularization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCharacteristics of rat LDOs\u003c/h3\u003e\n\u003cp\u003eRat bile duct fragments were isolated after enzymatic digestion, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and LDOs proliferated rapidly when cultured in expansion medium (EM) embedded in Matrigel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). LDOs in expansion culture were analyzed using immunofluorescence to determine the expression of markers specific to cholangiocytes and hepatocytes. Compared to native liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), Immunofluorescence analyses revealed that the LDOs expressed cholangiocyte lineage markers KRT19 and SOX9, while the hepatocyte markers ALB and Hnf4a were absent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings indicate that LDOs cultured under expansion conditions exhibited cholangiocytic characteristics.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eBile canalicular formation in recellularized liver with PHs\u003c/h3\u003e\n\u003cp\u003eFreshly isolated rat PHs (5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells) were injected via the BD of the scaffolds, followed by perfusion culture via the PV for 2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHistological analysis demonstrated appropriate distribution of PHs within the parenchymal space, with some cells engrafted and adherent to each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Immunofluorescence revealed MRP2 and ZO-1 expression on the membrane side of viable hepatocytes, showing patterns similar to those of native liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Electron microscopy confirmed bile canalicular structures between hepatocytes, characterized as a space with microvilli surrounded by tight junctions, similar to the native liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003eReconstruction of biliary structure in recellularized liver with PHs and LDOs\u003c/h3\u003e\n\u003cp\u003eTo reconstruct biliary structure, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e LDO-derived cells were injected via the BD and perfused with EM via the PV for 5 days. Then, freshly isolated rat PHs (5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells) were injected via the BD, followed by perfusion culture with co-culture medium via the PV for 2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHistological analyses revealed the distribution of cells engrafted into the BD lumen and parenchymal space. Immunofluorescence confirmed that GFP-positive cells corresponded to KRT19-positive cells and were distinct from albumin-positive cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicate that LDOs reconstructed the BDs and PHs were engrafted in the parenchyma, maintaining their respective characteristics.\u003c/p\u003e\u003cp\u003eIn some cell clusters, the cells engrafted into the BD and parenchyma adhered to each other. In these areas, MRP2 expression in the recellularized PHs was co-localized with KRT19-positive BDs, resembling native liver architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003eExcretion of bile acids into recellularized BDs\u003c/h3\u003e\n\u003cp\u003eDuring the 2-day perfusion culture after recellularization of PHs and LDOs, approximately 30\u0026ndash;50\u0026micro;L of drainage fluid from the BD was collected using a syringe connected to the culture dish (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eELISA for the total bile acids in the biliary drainage and culture medium were performed. In the three samples that exhibited bile canaliculi with MRP2 expression close to the BDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the total bile acid concentrations in the biliary drainage tended to be higher than in the culture medium (6.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43 vs 2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29nmol/10\u003csup\u003e6\u003c/sup\u003ecells, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.100; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In contrast, among the five specimens lacking bile canaliculi in proximity to bile ducts, no significant difference in concentration was observed (3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80 vs 5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93nmol/10\u003csup\u003e6\u003c/sup\u003ecells, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.310) (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, rat PHs injected into decellularized liver scaffolds adhered to each other, repolarized, and formed bile canaliculi. Co-recellularization with rat PHs and LDOs enabled the reconstruction of biliary structures, with BDs repopulated by LDOs and bile canaliculi formed by PHs within certain cell clusters. In samples where the biliary structure was successfully reconstructed, the total bile acid concentration in the biliary drainage from the recellularized liver tended to be higher than that in the perfusing culture medium. These findings suggest that a 3D biliary excretion system can be reconstructed in a bioengineered liver using decellularized scaffolds recellularized with rat PHs and LDOs.\u003c/p\u003e\u003cp\u003eOver the past decade, significant advancements have been made in the development of decellularized liver-based bioengineered livers using various recellularized cell sources for clinical applications. Takeishi et al. reported that hepatocytes, cholangiocytes, and vascular endothelial cells derived from human induced pluripotent stem cells successfully recellularized rat liver scaffolds and could be transplanted into immunocompromised rats [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Human-specific albumin and A1AT were identified in the serum using ELISA 4 days after transplantation, indicating graft functionality in vivo. In a more clinically relevant study, Higashi et al. reported heterotopic transplantation of porcine recellularized liver grafts with PHs and vascular endothelial cells into porcine liver failure models [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Angiography and contrast-enhanced CT on postoperative day 28 confirmed graft perfusion, and serum markers such as total bilirubin, AST, ALT, ALP, and NH₃ were lower in the transplant group than in the control group at most postoperative time points. Despite these developments towards the adoption of bioengineered livers in clinical settings, the biliary system has not been sufficiently addressed.\u003c/p\u003e\u003cp\u003eIn this study, we first focused on how the recellularized hepatocytes engrafted onto the scaffold. We demonstrated that rat PHs can repolarize and form bile canaliculi, as shown by ZO-1 and MRP2 expression and transmission electron microscopy at the parenchymal space. These results indicate that the extracellular matrix of the liver scaffold facilitates hepatocyte engraftment and contributes to the formation of bile canaliculi. To the best of our knowledge, this is the first study to demonstrate the reconstruction of bile canalicular structures and their distribution within a recellularized liver.\u003c/p\u003e\u003cp\u003eTwo-dimensional reconstruction of a bile excretion system has been reported. Tanimizu et al. co-cultured mouse small hepatocytes and primary cholangiocytes in a two-dimensional culture environment, enabling the development of cell populations, which retained the functional connections of hepatocytes and cholangiocytes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In their study, bilirubin and fluorescein-labeled bile acid were absorbed by hepatocytes, excreted into bile canaliculi, and accumulated in the biliary system. Huang et al. used chemically induced liver progenitors to engineer an integrated tubule-hepatocyte tissue [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Chemically induced liver progenitors were induced from mature hepatocytes, differentiated into biliary epithelial cells, and formed biliary duct-like structures. Rat PHs were then plated onto tubular biliary-duct-like structures and cultured for 5 days in two-dimensional culture conditions, resulting in the formation of integrated tubule-hepatocyte tissue with functional interaction between hepatocytes and chemically induced liver progenitor-derived biliary-duct-like structures.\u003c/p\u003e\u003cp\u003eThree-dimensional reconstruction of a bile excretion system in bioengineered livers could significantly contribute not only to the adoption of bioengineered livers in transplantation medicine but also to the field of drug development. Several studies have attempted 3D reconstruction of the biliary system in decellularized liver scaffolds using recellularization techniques [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; however, they did not demonstrate detailed biliary structure, bile canaliculi, and BD, or data suggesting functional reconstruction of the biliary excretion system.\u003c/p\u003e\u003cp\u003eIn this study, we used LDOs as recellularization sources for decellularized BDs. We previously reported that LDOs have cholangiocyte characteristics and can efficiently repopulate BDs of the liver scaffold [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. An advantage of using LDOs is their genetic stability during long-term expansion, which allows a large cell supply. Another advantage is the potential to overcome immunogenicity, which is a major challenge in transplantation medicine, as LDOs can be isolated from human specimens for BD recellularization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe demonstrated that serial recellularization of rat LDOs and PHs successfully generated tissues wherein the BDs repopulated with LDOs were in close proximity to bile canaliculi formed by PHs. A schematic diagram of the reconstructed bile excretion system in the recellularized liver is provided in Supplementary Figure S2. Hepatocytes spread into the parenchymal space, while reconstructed BDs connected with hepatocytes in specific areas. The arrangement of bile canaliculi and BDs closely resembled the native liver, suggesting the reconstruction of the biliary system.\u003c/p\u003e\u003cp\u003eThere have been no reports on the reconstruction of bioengineered livers with a functional bile excretion system. In vivo, bile is produced by hepatocytes, secreted into bile canaliculi, transported through BDs, and discharged into the intestinal tract. Bile acid concentration is higher in bile than in the bloodstream; thus, if the bile excretion pathway is successfully reconstructed, the concentration of bile acids in BD effluents should be higher than in the circulating medium. Our experiments demonstrated that only specimens where bile canaliculi formed in proximity to BDs showed a tendency toward higher bile acid concentrations in the biliary drainage. To our knowledge, this is the first study to evaluate bile excretion function in bioengineered livers generated from decellularized scaffolds.\u003c/p\u003e\u003cp\u003eA limitation of this study is that real-time bile flow in recellularized livers was not assessed; therefore, future studies should address this using fluorescent bile acid excretion assays and by performing \u003cem\u003eex vivo\u003c/em\u003e and/or \u003cem\u003ein vivo\u003c/em\u003e blood perfusion experiments. Additionally, the present study does not establish a method to efficiently generate samples in which bile canaliculi and bile ducts are located in close proximity, indicating that further refinement of the approach is required. Ideally, if the bile excretion system in recellularized livers can be successfully reconstructed using autologous hepatocyte sources, it would significantly enhance the clinical potential of bioengineered livers.\u003c/p\u003e\u003cp\u003eIn summary, our findings indicate the potential for reconstructing a 3D biliary structure in bioengineered livers generated through recellularization of liver scaffolds with rat PHs and LDOs. Bile excretion function was also suggested, representing a significant advance toward the realization of bioengineered livers.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eLewis rats (200\u0026ndash;300 g; SLC, Hamamatsu, Japan) were used to prepare the 3D liver scaffolds, PHs, and LDOs. Rats were housed under controlled temperature and humidity conditions with a 12-hour light/dark cycle and were provided standard chow and water ad libitum at the Kyoto University animal facility. Rats used for liver procurement were euthanized by exsanguination under deep isoflurane anesthesia, in accordance with institutional guidelines and approved protocols. All animal experiments were approved by the Kyoto University Animal Experimentation Committee under protocols MedKyo22166 (valid from April 7, 2017 to March 31, 2023) and 24184 (from April 1, 2023 to March 31, 2025), and were performed in accordance with the Kyoto University Animal Protection Guidelines. The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLiver procurement and decellularization\u003c/h2\u003e\u003cp\u003eLiver procurement and decellularization were conducted as previously reported [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. After cannulation of the bile duct with a 24-gauge catheter and the portal vein with a 20-gauge catheter, livers were flushed with PBS. The organs were frozen at \u0026minus;\u0026thinsp;80\u0026deg;C, thawed overnight at 4\u0026deg;C, and then subjected to enzymatic perfusion with 0.25% (w/v) trypsin\u0026ndash;1 mmol/L EDTA\u0026ndash;4Na containing phenol red (Wako, Osaka, Japan) at 37\u0026deg;C for 1 h. Detergent perfusion was subsequently performed using 0.1% polyoxyethylene octyl phenyl ether (Wako) and 0.05% EDTA (Sigma, St. Louis, USA) at 0.5 mL/min for 48 h. Decellularized grafts were sterilized by perfusion with 0.1% peracetic acid (Sigma-Aldrich) for 2 h, followed by extensive PBS washing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of cell sources\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003ePHs\u003c/h2\u003e\u003cp\u003eRat PHs were isolated using a two-step collagenase perfusion technique, as previously described [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Lewis rats were anesthetized with isoflurane (Wako), and the portal vein was cannulated with a 25-gauge needle. After transection of the suprahepatic and infrahepatic inferior vena cava, the liver was perfused with 50 mL of Ca\u0026sup2;⁺-free HBSS containing 0.5 mM EGTA (Wako) and 2 U/mL heparin (Mochida, Tokyo, Japan) for 5 min, followed by 50 mL of a collagenase-based digestion buffer at 37\u0026deg;C for 10 min. The digestion buffer consisted of 0.15% dispase II (Sanko Junyaku, Tokyo, Japan), 0.15% collagenase type II (Gibco, Palo Alto, CA, USA), 0.1 mg/mL DNase I (Sigma, St. Louis, USA), 2 U/mL heparin, and physiological salts and glucose (NaCl 150 mmol/L, KCl 5.4 mmol/L, NaHPO₄ 0.34 mmol/L, MgSO₄ 0.1 mmol/L, CaCl₂ 5.0 mmol/L, NaHCO₃ 4.2 mmol/L, glucose 5.6 mmol/L, and HEPES 10 mmol/L; Wako). After perfusion, the liver was excised, gently agitated to release cells, and passed through a 100 \u0026micro;m mesh filter (BD Falcon, Franklin Lakes, NJ, USA). The cell suspension was washed three times by centrifugation at 50\u0026times;g for 3 min at 4\u0026deg;C. PHs were used immediately for recellularization.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGreen fluorescent protein (GFP)-expressing LDOs\u003c/h2\u003e\u003cp\u003eRat liver ductal organoids (LDOs) were established based on previously described protocols in mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], with modifications. The left lateral liver lobe from Lewis rats was excised, cut into 2\u0026ndash;3 mm pieces, and further fragmented by sequential passage through 18-, 20-, 22-, and 23-gauge needles. Tissue fragments were then enzymatically digested in DMEM (Wako, Osaka, Japan) containing 2.6 \u0026micro;U/mL Liberase DH (Roche, Basel, Switzerland) and 0.1 mg/mL DNase I (Sigma-Aldrich, St. Louis, MO) at 37\u0026deg;C for 10 min. The resulting suspension was filtered through 100 \u0026micro;m and 40 \u0026micro;m meshes, and collected clusters were pelleted by centrifugation at 440 \u0026times; g for 3 min. Approximately 20\u0026ndash;30 clusters were resuspended in 7 \u0026micro;L of Matrigel-GFR (Corning, NY) and seeded on non-treated culture plates (IWAKI, Shizuoka, Japan). After polymerization at 37\u0026deg;C for 10 min, embedded clusters were cultured in expansion medium (EM) for 5\u0026ndash;7 days until organoid formation. Organoids were passaged every 4\u0026ndash;6 days using TrypLE Express (Invitrogen, Carlsbad, CA, USA) at 37\u0026deg;C for 15 min, and re-embedded in Matrigel-GFR for further expansion or recellularization.\u003c/p\u003e\u003cp\u003eFor genetic labeling, a GFP-expressing lentiviral vector (pLX304-GFP) was constructed by transferring the GFP coding sequence from pALB-GFP (#55759; Addgene) to pENTR4 (#17424; Addgene), followed by Gateway cloning into pLX304 (#25890; Addgene). Lentiviral particles were produced in HEK293FT cells co-transfected with pLX304-GFP, psPAX2 (#12260; Addgene), and pMD2.G (#12259; Addgene) using X-tremeGENE\u0026trade; HD (Roche), according to the manufacturer\u0026rsquo;s instructions. Viral supernatants were collected at 48 and 72 h after transfection and filtered through 0.45 \u0026micro;m PVDF membranes (Merck Millipore).\u003c/p\u003e\u003cp\u003eFor transduction, LDOs were dissociated into single cells and incubated with the viral supernatant supplemented with 10 \u0026micro;M Y-27632 (LC Laboratories, Woburn, MA), 1 mM N-acetyl-L-cysteine (Wako), and 10 \u0026micro;g/mL polybrene (Sigma). The cell\u0026ndash;virus mixture was transferred to untreated plates and centrifuged at 2000 rpm for 1 h at room temperature. After centrifugation, the supernatant was removed, and the cells were embedded in Matrigel-GFR and cultured in EM. Organoids were subsequently expanded and selected with blasticidin (Wako).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eRecellularization and perfusion culture of rat whole liver\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003ePerfusion culture of recellularized liver with only PHs\u003c/h2\u003e\u003cp\u003eRecellularization was performed based on our previously reported method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], with modifications. A total of 5.0 \u0026times; 10⁷ primary hepatocytes were resuspended in 30 mL of HCM\u0026trade; medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and infused into the liver scaffold via the bile duct at a rate of 1 mL/min. Following cell injection, the graft was maintained under static conditions at 37\u0026deg;C for 3 h to allow cell attachment. The recellularized liver was then connected to a perfusion circuit through the portal vein cannula and continuously perfused with HCM\u003csup\u003eTM\u003c/sup\u003e-based medium containing 10% FBS, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin using a peristaltic pump (Perista pump, ATTA, Japan) at 0.7\u0026ndash;1.0 mL/min at 37\u0026deg;C. The perfusate exiting through the inferior vena cava was collected and recirculated within the system. Perfusion culture was maintained for 48 h.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003ePerfusion culture of recellularized liver with LDOs and PHs\u003c/h2\u003e\u003cp\u003eSingle-cell suspensions of LDOs (3\u0026ndash;5 \u0026times; 10⁶ cells) were prepared in 5 mL of EM and infused into the liver scaffold through the BD at a flow rate of 1 mL/min. The graft was then maintained under static conditions in EM for 3 h before being connected to the perfusion circuit as described above. The culture medium was replaced every other day, and perfusion culture was continued for 5 days. Subsequently, 5.0 \u0026times; 10⁷ rat primary hepatocytes resuspended in 30 mL of co-culture medium (Supplementary Table S2) were infused via the BD. After static incubation for 3 h, perfusion culture was resumed with co-culture medium and maintained for an additional 48 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCollection of drainage fluids from the BD\u003c/h2\u003e\u003cp\u003eDuring perfusion culture, the BD of the scaffolds recellularized with PH and LDO was connected to a syringe via a culture dish, and negative pressure was applied by pulling the plunger of the 1 mL syringe and securing it with a thread.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eHistological analysis\u003c/h2\u003e\u003cp\u003eOrganoids were isolated from Matrigel, re-embedded in Cellmatrix Type I-A (Nitta Gelatin), and fixed overnight in 10% formalin (Wako) at room temperature. Recellularized liver grafts were fixed in 4% paraformaldehyde (Wako) for 24 h at 4\u0026deg;C and subsequently embedded in paraffin.\u003c/p\u003e\u003cp\u003eParaffin blocks were sectioned at 4 \u0026micro;m, deparaffinized, rehydrated, and subjected to hematoxylin and eosin staining or immunohistochemistry. For immunostaining, antigen retrieval was performed by autoclaving at 121\u0026deg;C for 15 min. Sections were blocked with PBS containing 10% donkey or goat serum and 0.1% Triton X-100 (Nacalai Tesque, Kyoto, Japan), followed by overnight incubation at 4\u0026deg;C with primary antibodies diluted in PBS supplemented with 5% donkey or goat serum and 0.1% Triton X-100. Secondary antibodies were applied for 1 h at room temperature.\u003c/p\u003e\u003cp\u003eNuclei were counterstained and mounted with ProLong\u0026trade; Gold Antifade Mountant containing DAPI (Invitrogen), and images were acquired using an Olympus BX50F4 microscope (Olympus, Tokyo, Japan). For each sample, five sections representing all hepatic lobes were analyzed to ensure unbiased histological evaluation. The list of antibodies used is provided in Supplementary Table S3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eElectron microscopy\u003c/h2\u003e\u003cp\u003eElectron microscopy was carried out as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Fresh and decellularized livers from Lewis rats were fixed overnight at 4\u0026deg;C in PBS containing 2% glutaraldehyde (Nacalai Tesque) and 4% paraformaldehyde. Fixed tissues were microdissected, dehydrated through a graded ethanol series (50, 60, 70, 80, 90, 99, and 100%), and immersed in t-butanol. Samples were then frozen at \u0026minus;\u0026thinsp;20\u0026deg;C, followed by sublimation. After sputter coating with a platinum\u0026ndash;palladium alloy using an ion coater (JBC-3000FC, JEOL, Japan), specimens were examined with a scanning electron microscope (JSM-7900F, JEOL).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA) of total bile acids\u003c/h2\u003e\u003cp\u003eAfter 48 h of perfusion culture of scaffolds recellularized with PHs and LDOs, culture medium and BD drainage fluids were collected. Total bile acid concentrations in both samples were measured using a Total Bile Acids Assay Kit (Cell Biolabs, San Diego, CA, USA) via ELISA. Background bile acid concentrations derived from FBS were measured in medium alone and subtracted from the values obtained for all samples. Bile acid concentrations were normalized to the number of engrafted hepatocytes (per 10\u003csup\u003e6\u003c/sup\u003e cells).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eStatistical methods\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Statistical analyses were performed using R version 4.1.0 (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Total bile acid concentrations were compared using an unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e3D, three-dimensional; BD, bile duct; ELISA, enzyme-linked immunosorbent assay; EM, expansion medium; ESLD, end-stage liver disease; GFP, green fluorescent protein; LDO, liver ductal organoids; PH, primary hepatocytes; PV, portal vein.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University, for technical assistance with the analysis of electron microscopy data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI [grant numbers 22K08689, 24KJ1416].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u0026nbsp;\u003c/strong\u003eKen Fukumitsu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u0026nbsp;\u003c/strong\u003eHiroshi Horie,\u0026nbsp;Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition\u003c/strong\u003e: Hiroshi Horie, Ken Fukumitsu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation\u003c/strong\u003e: Hiroshi Horie, Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hidenobu Kojima, Satoshi Ogiso, Elena Yukie Uebayashi, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology\u003c/strong\u003e: Hiroshi Horie, Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hidenobu Kojima, Satoshi Ogiso, Elena Yukie Uebayashi, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration\u003c/strong\u003e: Ken Fukumitsu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003e Hiroshi Horie, Ken Fukumitsu, Masahiro Inoue\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision\u003c/strong\u003e: Masahiro Inoue, Takamichi Ishii, Etsuro Hatano\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting - original graft:\u0026nbsp;\u003c/strong\u003eHiroshi Horie, Ken Fukumitsu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting - review and editing:\u0026nbsp;\u003c/strong\u003eSatoshi Ogiso, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, K.F., upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAsrani, S.K., et al., \u003cem\u003eBurden of liver diseases in the world.\u003c/em\u003e J Hepatol, 2019. \u003cstrong\u003e70\u003c/strong\u003e(1): p. 151-171.\u003c/li\u003e\n\u003cli\u003eNicolas, C.T., et al., \u003cem\u003eConcise Review: Liver Regenerative Medicine: From Hepatocyte Transplantation to Bioartificial Livers and Bioengineered Grafts.\u003c/em\u003e Stem Cells, 2017. \u003cstrong\u003e35\u003c/strong\u003e(1): p. 42-50.\u003c/li\u003e\n\u003cli\u003eBhatia, S.N., et al., \u003cem\u003eCell and tissue engineering for liver disease.\u003c/em\u003e Sci Transl Med, 2014. \u003cstrong\u003e6\u003c/strong\u003e(245): p. 245sr2.\u003c/li\u003e\n\u003cli\u003eUygun, B.E., et al., \u003cem\u003eOrgan reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.\u003c/em\u003e Nat Med, 2010. \u003cstrong\u003e16\u003c/strong\u003e(7): p. 814-20.\u003c/li\u003e\n\u003cli\u003eSoto-Gutierrez, A., et al., \u003cem\u003eA whole-organ regenerative medicine approach for liver replacement.\u003c/em\u003e Tissue Eng Part C Methods, 2011. \u003cstrong\u003e17\u003c/strong\u003e(6): p. 677-86.\u003c/li\u003e\n\u003cli\u003eBaptista, P.M., et al., \u003cem\u003eThe use of whole organ decellularization for the generation of a vascularized liver organoid.\u003c/em\u003e Hepatology, 2011. \u003cstrong\u003e53\u003c/strong\u003e(2): p. 604-17.\u003c/li\u003e\n\u003cli\u003eChen, C., et al., \u003cem\u003eHepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers.\u003c/em\u003e Biotechnol Bioeng, 2018. \u003cstrong\u003e115\u003c/strong\u003e(11): p. 2807-2816.\u003c/li\u003e\n\u003cli\u003eKojima, H., \u003cem\u003eEstablishment of practical recellularized liver graft for blood perfusion using primary rat hepatocytes and liver sinusoidal endothelial cells.\u003c/em\u003e American journal of transplantation, 2018. \u003cstrong\u003e18\u003c/strong\u003e(6): p. 1351-1359.\u003c/li\u003e\n\u003cli\u003eMinami, T., et al., \u003cem\u003eNovel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold.\u003c/em\u003e Regen Ther, 2019. \u003cstrong\u003e10\u003c/strong\u003e: p. 127-133.\u003c/li\u003e\n\u003cli\u003eOgiso, S., et al., \u003cem\u003eEfficient recellularisation of decellularised whole-liver grafts using biliary tree and foetal hepatocytes.\u003c/em\u003e Sci Rep, 2016. \u003cstrong\u003e6\u003c/strong\u003e: p. 35887.\u003c/li\u003e\n\u003cli\u003eTomofuji, K., et al., \u003cem\u003eLiver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts.\u003c/em\u003e Biomaterials, 2022. \u003cstrong\u003e287\u003c/strong\u003e: p. 121614.\u003c/li\u003e\n\u003cli\u003eTakeishi, K., et al., \u003cem\u003eAssembly and Function of a Bioengineered Human Liver for Transplantation Generated Solely from Induced Pluripotent Stem Cells.\u003c/em\u003e Cell Rep, 2020. \u003cstrong\u003e31\u003c/strong\u003e(9): p. 107711.\u003c/li\u003e\n\u003cli\u003eLewis, P.L., et al., \u003cem\u003eComplex bile duct network formation within liver decellularized extracellular matrix hydrogels.\u003c/em\u003e Sci Rep, 2018. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 12220.\u003c/li\u003e\n\u003cli\u003eChen, Y., et al., \u003cem\u003eRepopulation of intrahepatic bile ducts in engineered rat liver grafts.\u003c/em\u003e Technology (Singap World Sci), 2019. \u003cstrong\u003e7\u003c/strong\u003e(1-2): p. 46-55.\u003c/li\u003e\n\u003cli\u003eKo, I.K., et al., \u003cem\u003eBioengineered transplantable porcine livers with re-endothelialized vasculature.\u003c/em\u003e Biomaterials, 2015. \u003cstrong\u003e40\u003c/strong\u003e: p. 72-9.\u003c/li\u003e\n\u003cli\u003eZhou, P., et al., \u003cem\u003eDecellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.\u003c/em\u003e Artif Organs, 2016. \u003cstrong\u003e40\u003c/strong\u003e(3): p. E25-38.\u003c/li\u003e\n\u003cli\u003eTanimizu, N., et al., \u003cem\u003eGeneration of functional liver organoids on combining hepatocytes and cholangiocytes with hepatobiliary connections ex vivo.\u003c/em\u003e Nat Commun, 2021. \u003cstrong\u003e12\u003c/strong\u003e(1): p. 3390.\u003c/li\u003e\n\u003cli\u003eHuang, Y., et al., \u003cem\u003eBioengineering of a CLiP-derived tubular biliary-duct-like structure for bile transport in vitro.\u003c/em\u003e Biotechnol Bioeng, 2021. \u003cstrong\u003e118\u003c/strong\u003e(7): p. 2572-2584.\u003c/li\u003e\n\u003cli\u003eHigashi, H., et al., \u003cem\u003eTransplantation of bioengineered liver capable of extended function in a preclinical liver failure model.\u003c/em\u003e Am J Transplant, 2022. \u003cstrong\u003e22\u003c/strong\u003e(3): p. 731-744.\u003c/li\u003e\n\u003cli\u003eAnderson, B.D., et al., \u003cem\u003eFunctional characterization of a bioengineered liver after heterotopic implantation in pigs.\u003c/em\u003e Commun Biol, 2021. \u003cstrong\u003e4\u003c/strong\u003e(1): p. 1157.\u003c/li\u003e\n\u003cli\u003eHirukawa, K., et al., \u003cem\u003eNovel approach for reconstruction of the three-dimensional biliary system in decellularized liver scaffold using hepatocyte progenitors.\u003c/em\u003e PLoS One, 2024. \u003cstrong\u003e19\u003c/strong\u003e(2): p. e0297285.\u003c/li\u003e\n\u003cli\u003eChen, J., et al., \u003cem\u003eGeneration and metabolomic characterization of functional ductal organoids with biliary tree networks in decellularized liver scaffolds.\u003c/em\u003e Bioact Mater, 2023. \u003cstrong\u003e26\u003c/strong\u003e: p. 452-464.\u003c/li\u003e\n\u003cli\u003eKr\u0026uuml;ger, M., et al., \u003cem\u003eHigh level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.\u003c/em\u003e J Cell Mol Med, 2022. \u003cstrong\u003e26\u003c/strong\u003e(19): p. 4949-4958.\u003c/li\u003e\n\u003cli\u003eWillemse, J., et al., \u003cem\u003eScaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish.\u003c/em\u003e Biotechnol Bioeng, 2021. \u003cstrong\u003e118\u003c/strong\u003e(2): p. 836-851.\u003c/li\u003e\n\u003cli\u003eHorie, H., et al., \u003cem\u003eAntithrombotic Revascularization Strategy of Bioengineered Liver Using a Biomimetic Polymer.\u003c/em\u003e Tissue Eng Part A, 2025. \u003cstrong\u003e31\u003c/strong\u003e(11-12): p. 433-441.\u003c/li\u003e\n\u003cli\u003eKita, S., et al., \u003cem\u003eThe Protective Effect of Transplanting Liver Cells Into the Mesentery on the Rescue of Acute Liver Failure After Massive Hepatectomy.\u003c/em\u003e Cell Transplant, 2016. \u003cstrong\u003e25\u003c/strong\u003e(8): p. 1547-59.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Liver regeneration, tissue engineering, liver ductal organoids, hepatocytes, bioartificial organs","lastPublishedDoi":"10.21203/rs.3.rs-7659575/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7659575/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBioengineered livers are potential alternatives for liver transplantation in patients with end-stage liver disease. Liver scaffolds engineered through decellularization techniques have been developed for clinical applications; however, reconstruction of a functional biliary system remains a challenge. This study aimed to establish a three-dimensional biliary system in bioengineered livers through recellularization of rat primary hepatocytes (PHs) and liver ductal organoids (LDOs).\u003cstrong\u003e \u003c/strong\u003eDecellularized rat liver scaffolds were recellularized using rat PHs and green fluorescent protein (GFP)-expressing LDOs. Dissociated LDOs were injected via the bile duct and cultured for 5 days using a perfusion device, followed by PH injection and 2 days of culture. During co-culture, biliary drainage fluid and culture medium were collected to compare total bile acid concentrations using enzyme-linked immunosorbent assay. Histological and immunofluorescence analyses were performed after 7 days of perfusion culture.\u003cstrong\u003e \u003c/strong\u003eHistological analyses confirmed the engraftment of GFP-expressing LDOs into bile ducts and PHs into the parenchymal space. Engrafted PHs expressed ZO-1 and MRP2, forming bile canaliculi. In specific regions, MRP2-positive PHs and KRT19-positive LDO-repopulated cells adhered to each other, resembling the native liver structure. In the samples exhibiting such structures, total bile acid concentrations in the biliary drainage fluid tended to be higher than in the culture medium.This study suggested that a three-dimensional functional biliary system could be reconstructed in bioengineered livers. These findings represent a significant step toward the realization of bioengineered livers using decellularization and recellularization techniques.\u003c/p\u003e","manuscriptTitle":"Three-dimensional reconstruction of a biliary system in a bioengineered liver using decellularized scaffold","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-20 08:16:05","doi":"10.21203/rs.3.rs-7659575/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-09T06:55:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T07:34:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T11:40:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-01T01:30:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T19:57:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47962252102775480371690615436501196402","date":"2025-11-17T03:15:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246699507806724676130051708458271699015","date":"2025-11-13T08:17:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38463858138636968172443936432916804647","date":"2025-11-13T00:08:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300108204424501033342487785914655662660","date":"2025-11-11T05:40:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-10T22:39:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-10T05:05:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-07T05:06:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-01T04:28:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-01T04:25:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d3fbda7-ce33-4757-a7f6-5493374328c4","owner":[],"postedDate":"November 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58079626,"name":"Biological sciences/Biological techniques"},{"id":58079627,"name":"Biological sciences/Biotechnology"},{"id":58079628,"name":"Health sciences/Gastroenterology"},{"id":58079629,"name":"Health sciences/Medical research"},{"id":58079630,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-02-16T15:59:40+00:00","versionOfRecord":{"articleIdentity":"rs-7659575","link":"https://doi.org/10.1038/s41598-026-39175-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-10 15:56:56","publishedOnDateReadable":"February 10th, 2026"},"versionCreatedAt":"2025-11-20 08:16:05","video":"","vorDoi":"10.1038/s41598-026-39175-2","vorDoiUrl":"https://doi.org/10.1038/s41598-026-39175-2","workflowStages":[]},"version":"v1","identity":"rs-7659575","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7659575","identity":"rs-7659575","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}