An In Vitro Whole-Organ Liver Engineering for Testing of Genetic Therapies

Summary Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The in vitro whole-organ “Bioreactor grown Artificial Liver Model” (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.


INTRODUCTION
The liver accomplishes at least 500 vital functions ranging from bile production and protein synthesis to removal of blood toxins (Ehrlich et al., 2019). Liver transplantation is the only therapeutic option for a small proportion of monogenic liver-based disorders but is associated with significant morbidity and mortality, is limited by organ availability, and requires lifelong immune suppression. Treatments of liver diseases are complex, significantly impair patients' quality of life, and do not achieve perfect outcomes (Kriegermeier and Green, 2020;Martinelli et al., 2018).
Hence, liver-directed gene therapy, which consists of delivering a nucleic acid to compensate for a dysfunctional gene, is an attractive alternative (Baruteau et al., 2017). Different approaches that can be employed to modify endogenous genes include gene addition, gene editing, RNA-based therapies, and others. There has been a steady increase in liver-directed gene therapy clinical trials in recent years.
To ensure the success of gene therapy, the best delivery method and a vector that produces sufficient protein expression in target cells need to be selected (van Haasteren et al., 2018). Thus, the use of a model able to address these obstacles is essential.
Current in vitro liver models employ immortalized cell lines, like HepG2 or HepaRG, or primary human hepatocytes (PHHs) grown as a monolayer. These models, however, are of limited use owing to incomplete functional capacity (HepG2 and HepaRG cells), donor-to-donor variation, and rapid de-differentiation (PHHs). Two-dimensional (2D) cell cultures are grown at about 1% of normal tissues densities, which impairs intracellular signaling. To address these shortcomings three-dimensional (3D) in vitro models have been developed such as liver precision-cut tissue slices (PCTS), liver-on-a-chip microfluidic systems, and liver organoids. However, PCTS have only a short-term survival (Collins et al., 2019), whereas liver-on-chip microfluidic systems and organoids do not reflect the 3D liver architecture and lack natural extracellular matrix (ECM) and vascularization, essential for nutrient and oxygen exchange (Akbari et al., 2019).
Rodent disease models offer many advantages over in vitro liver models; however, the physiological and genomic interspecies differences pose limitations in the representation of the disease phenotypes (Mariotti et al., 2018;Martignoni et al., 2006) and vector targeting. Recombinant AAV8 vector was used in the first successful gene therapy clinical trial, which targeted hepatocytes (Nathwani et al., 2014). A small increase (achieving <10% of normal) in the plasma circulating factor IX, secreted by the liver, was sufficient to improve patients' phenotype. Much higher FIX levels were seen in the preclinical studies likely owing to the differences in hepatocyte transduction by AAV8 between humans and mice (Manno et al., 2006;Lisowski et al., 2014). Humanized FRG mice (where mouse liver is partially repopulated by human hepatocytes) is a better model to study human hepatocyte vector transduction (Strom et al., 2010); however, they are extremely resource intensive and require patient-specific hepatocytes in order to demonstrate disease phenotype.
Here we demonstrate testing of viral gene therapy vectors in an in vitro whole-organ ''Bioreactor grown Artificial Liver Model'' (BALM) that employs bioreactor for long-term 3D culture of human induced pluripotent stem cells (hiPSCs)-derived hepatocyte-like cells (hiHEPs). hiPSCs can provide an unlimited source of patient-derived cells, which can be differentiated toward hepatocyte lineage. BALM uses mouse decellularized liver scaffolds as growth support, with a preserved extracellular matrix (ECM) and 3D structure previously shown to promote a faster maturation of hiHEPs (Lorvellec et al., 2017).
Bioreactor provides controlled and dynamic culture conditions and is the solution to the development of 3D organ models. The concept of using bioreactor-engineered whole-organ systems could overcome some of the current caveats of in vitro and in vivo modeling and improves the chance of more accurate preclinical therapy testing outcomes.

Generation of BALM
The mouse livers were decellularized via cannulation of the portal vein (PV) by detergent-enzymatic treatment (DET), which preserves the natural ECM and the vascular network (Maghsoudlou et al., 2016;Mazza et al., 2017 ) ( Figure S1). The hiPSCs line previously used to generate hiHEPs (Song et al., 2009;Yusa et al., 2011) was differentiated toward definitive endoderm-like cells (DECs). DECs harvested at day 6 of differentiation were injected into multiple locations of the parenchyma of individual lobes of the mouse decellularized liver scaffolds. DEC differentiation toward hepatocytes was then continued in the bioreactor with the hepatic specification stage from day 7 to day 11 followed by the hepatic maturation stages 1 and 2 (Figure 1A). The stage-specific media, 3D environment, and liver ECM of the scaffold were previously shown to promote faster maturation of hiHEPs (Lorvellec et al., 2017).
The seeded decellularized livers were placed into the chamber of the bioreactor and connected to the circuit via its catheter at the entrance of the chamber. The volume of the chamber is 50.2 cm 3 keeping the media to a minimum volume of 20 mL. Media circulation in a closed circuit was driven by a roller pump, which delivered flow perfusion through the vascular network. This system permits nutrients and oxygen to reach the whole scaffold and creates dynamic shear stress, important for orientation of polarized epithelial cells such as hepatocytes (Tharp and Weaver, 2018;You et al., 2019;Dash et al., 2013). Media changes and collection were facilitated by the three-way connector at the exit of the chamber. A bubble trap before the entrance of the chamber eliminated air bubbles to avoid embolization of the scaffold. The media was oxygenated using a 5% CO 2 /Air gas cylinder regulated by a flowmeter, and the gas was humidified in a water bottle, avoiding excess evaporation while an overflow bottle collected outflow humidity. The system was maintained at 37 C using a hot plate placed under the chamber and the two bottles. The temperature and pH were monitored in real time by using a sterile dip sensor. The transparent lid of the chamber allowed visual monitoring of the scaffold (Figures 1B and 1C). A three-way connector placed before the entrance of the chamber permitted the perfusion of compounds or gene therapy products. The bioreactor system provides a sterile and controlled environment for the culture of the 3D whole-organ system. No malfunctioning or contaminations occurred during the experiments. The pH monitoring reported maintenance of the culture in the range of 7-7.8 with fluctuation derived from media changes according to the protocol ( Figure 1D). BALM was maintained until day 27 of cell differentiation. 10  The mouse decellularized liver scaffolds generated by DET were visually evaluated for the quality of vascular perfusion by injection of colored media via the PV catheter. After seeding, injection sites were observed in each seeded lobe as opaque masses among transparent surrounding tissue. After 25 days of differentiation in BALMs, the whole tissue became opaque (Figure 2A).
DAPI and H&E staining of cryosections of the individually seeded liver lobes showed many cell nuclei, whereas in the control unseeded lobes only the ECM fibers were visible ( Figures 2B and 2C). hiPSC-derived cells successfully repopulated the scaffolds and appeared to be positioned along the scaffold fibers. We previously demonstrated that the amount of DNA left in the decellularized liver is negligible compared with the fresh liver tissue (0.093 and 1.068 mg/mg, respectively) (Lorvellec et al., 2017). Ki-67, a marker of proliferation was highly expressed at day 21 of differentiation, whereas there were only a few apoptotic cells as shown by cleaved caspase-3 immunofluorescence staining. Cell viability assay showed mitotically active cells in BALMs at days 20, 26, and 27 ( Figures 2D, S2, and S5).
Expression of characteristic hepatocyte markers was analyzed on days 25-27 of differentiation in BALMs. Immunofluorescence staining of cryosections of seeded lobes showed that a high number of cultured hi-HEPs expressed albumin (ALB), a mature cytoplasmic hepatocyte marker, whereas few cells expressed Figure 1. BALM Generation (A) hiPSCs differentiated to DECs, mouse livers decellularized using DET method. DECs injected into multiple locations in individual lobes on day 6 of differentiation. Then livers were placed into bioreactor and cultured under perfusion. Hepatic specification stage: day 7 to day 11 followed by hepatic maturation. During the maturation stage, viral vectors were perfused through the vasculature. BALMs were kept in culture till day 27. (B) Stand-alone bioreactor composed of a chamber with medium in a closed circuit with a roller pump. The medium is oxygenated by 5% CO 2 /Air gas cylinder regulated by a flowmeter. The gas is humidified in a water bottle while an overflow bottle collects outflow humidity. A bubble trap before the entrance of the chamber eliminates air bubbles. The system is maintained at 37 C using a hot plate under the chamber and the bottles.   -Holgersson, 2012;Xu et al., 2007). Interestingly, asialoglycoprotein receptor (ASGPR), which is normally localized at the sinusoidal membrane of the mature hepatocytes, was restricted to a part of the membrane in cells cultured in BALMs. Thus, taken together, these findings suggest cell differentiation toward mature hepatocytes ( Figures 3A, S3, and S5).
Synthesis of plasma proteins is one of the hepatocyte functions with ALB being the most abundant. ALB production increases as hepatocytes mature from fetal to the adult cell type. AFP expression, on the contrary, is high in the fetus but drops soon after birth (Elmaouhoub et al., 2007;Nayak and Mital, 1977).
To monitor in a non-invasive manner the secretion of hepatic proteins, we harvested the BALM media during the hepatocyte maturation stage 2 and used targeted proteomic liquid chromatography with tandem mass spectrometry (UPLC-LC/MS/MS) technique to measure tryptic peptides from ALB and AFP. As the number of cells alive at the time of measurement is unknown, we calculated the ratio ALB/AFP. We observed that the ALB/AFP ratio increases from d19 of differentiation till d25, with raw data suggesting that this is the result of overall increase in ALB and decrease in AFP ( Figure 3B and Table S1). In concordance with the microscopy results, the increased ratio would also point toward hiHEPs becoming more mature during BALM culture.
To investigate whether endothelial cells could repopulate BALM vasculature and whether BALM could sustain coculture of different cell types, we perfused human umbilical vein endothelial cells (HUVECs) through the PV catheter and cocultured them with DECs up to day 21 of differentiation. Immunofluorescence staining of cryosections of DECs seeded lobes showed that only cells in the vasculature expressed CD31, an endothelial marker. HUVECs were able to repopulate BALM's vasculature network and were still proliferating as well as hiHEPs ( Figures 3C, S3, and S5). We did not observe any detrimental effects of the HUVECS on hiHEPs differentiation.
BALM as a Tool to Evaluate Viral Vector-Based Gene Therapy We investigated whether BALM can be used to test the efficiency of AAV and LV vectors for human liver cell transduction. Both types of vectors were perfused through the vasculature, during the maturation stage 2 ( Figure 1A).   Figure 4A). The optimal concentration of 1 mM agonist was determined using hiHEPs in 2D culture ( Figure S4).
BALMs were perfused on day 19 for 48 h with LNT-LXR-Nluc/eGFP, then fresh media with 1 mM T0901317 agonist was added on day 21 for 96 h (till day 25 of differentiation). The NanoLuc luciferase reagent furimazine was administered by perfusion through the catheter (Nano-Glo Luciferase assay, Promega). Live bioluminescence was observed before and after addition of the substrate showing activation of the LNT-LXR-Nluc/eGFP reporter construct inside the seeded lobes of the scaffold with average RLU/scaffold ranging from 4 3 10 7 to 3.3 3 10 8 ph/s (Figures 4B and 4C and Table S2). Immunofluorescence staining of eGFP was performed on cryosections of individual lobes and showed GFP-positive cells in BALMs transduced for 2 days with LNT-LXR-Nluc/eGFP ( Figures 4D and S5).
The small NanoLuc luciferase is secreted into the media and therefore allows monitoring of luciferase activity by simply sampling the media from the bioreactor. Initially, PHHs were transduced for 24 h in 2D culture. Then fresh media with 1 mM T0901317 agonist was added for 48 h and the media was assayed for luciferase activity; 3.08 3 10 5 PHHs gave a fold change of 1.3 3 10 4 (Figures 4F and Table S2).
Thus, we showed that LV could transduce hiHEPs when delivered through the vasculature of the whole-organ liver model. Bioluminescence imaging performed on the day of harvest demonstrated that the cells were alive at that time and the LXR receptor was active in those cells. Furthermore, it also allowed us to locate the highest density of live cells within the scaffold.

DISCUSSION
Cell culture models are fundamental instruments for basic and translational research. Clinical trials frequently fail owing to the lack of direct translation from preclinical experiments into human studies (Manno et al., 2006;Pound and Ritskes-Hoitinga, 2018). The development of models that can better replicate human tissue complexity is needed.
The BALM system has several advantages over conventional in vitro models. It utilizes hiHEPs, which, once derived from disease-specific hiPSCs, could allow disease modeling and phenotype correction. The scaffolds derived from adult mice livers provide the ECM proteins and the 3D structure reproducing the in vivo liver environment and can be obtained from repurposed animals, and therefore, this approach is aligned with the 3Rs (replacement, reduction, and refinement) of animal research. The stand-alone bioreactor is independent of a cell culture incubator, allowing the device to be located on a standard laboratory benchtop. Different parameters can be easily adjusted in this controlled environment such as oxygen level, moving from normoxia to hypoxia; temperature; and pH, which can be monitored in real time. The latter is essential as it influences cell proliferation and cell volume; pH of the media below 6.8 or above 8 is detrimental to cell growth (Mackenzie et al., 1961). Furthermore, tight control of intracellular pH of hepatocytes is essential for its metabolic roles, including urea synthesis, glycolysis, and bile electrolyte secretion (Pollock, 1984;Strazzabosco and Boyer, 1996;Park et al., 1979). iScience Article The cannulation of the portal vein (PV) in our model allows delivery of oxygen, nutrients, and therapeutic products through the liver vasculature mimicking the natural conditions. Indeed the liver receives 25% of the cardiac output, mostly via the portal vein (van Haasteren et al., 2018). The advantage of having PV access allows modification of the flow in the vascular network using the roller pump controlling the fluid shear stress (Tresoldi et al., 2015). Moreover, we demonstrated that AAV and LV vectors can transduce the hiHEPs in BALM when delivered via the PV and can drive transgene expression. BALMs can also be used to investigate repeated delivery of therapeutics.
In contrast to BALM, most in vitro models including organoids and liver-on-chip microfluidic systems lack the extensive vasculature or sinusoids that are needed for nutrient exchange. The small dimensions of the microfluidic systems allow surface effects to dominate volume effects leading to a laminar flow with little mixing ( The development of functional assays using bioreactor media as well as the use of bioluminescent or fluorescent reporters allow non-invasive monitoring of cell viability and function. The BALM cultured hiHEPs were alive and functional on day 27 of differentiation evidenced by secreted ALB and AFP detection as well as the LXR reporter activity. The differentiation of hiPSC and the prolonged culture in the bioreactor allowed the delivery of the gene therapy products at different stages of human hepatocyte development and permitted its long-term study.
To improve BALMs, the number of DECs seeded on day 6 could be scaled up enhancing the overall distribution of the hepatocytes throughout the scaffold. Higher cell numbers would improve the development of the functional assays as most routinely used 2D assays have to be adapted to the challenges of the BALM system where the cells are less easily accessible to examination and their secreted products are highly diluted in the bioreactor media. Development of bioreactor-specific assays to study ureagenesis or gluconeogenesis would be highly beneficial to further characterize the functionality of hiHEPs.
Further improvements could benefit BALMs. Although coculture of hiHEPs and HUVECs seeded through the PV allowed a better mimicking of the liver environment (Huang et al., 2020), it has been very recently shown that also endothelial and biliary cell types could be derived from human iPSC and co-seeded with iPSC-derived hepatocytes in decellularized livers to form functional mini livers. The latter could be used for auxiliary transplantation in immunocompromised rats and remain functional for 4 days in vivo (Takeishi et al., 2020). Moreover, multiple cannulations of hepatic artery, hepatic veins, and bile duct would provide additional delivery routes for other liver cells and therapeutic compounds. This will require a combination of culture media that would allow co-culture of all liver cells (Ehrlich et al., 2019), but it may help to establish the metabolic zonation (Ehrlich et al., 2019), which would make this 3D whole organ even more representative of live conditions and facilitate testing of drug delivery to the whole-cell gamut.
In conclusion, the humanized whole-organ liver model BALM is a valuable tool that could replace some of the in vitro and in vivo therapeutic testing approaches generating high-value pre-clinical data.

Limitations of the Study
BALM has a few challenges to overcome linked to its 3D nature: it contains a small proportion of cells, not easily accessible, for a high amount of ECM, and their secreted products are highly diluted in the bioreactor media. Establishing a standard curve for cell viability assays was impaired owing to lack of cells with similar metabolism to the hiHEPs differentiated in BALMs and to 2D versus 3D differences: reagent's incubation time and dilution, percentage of hiHEPs and background level. Nevertheless, the presto blue assay could be used to show a relative effect of a particular condition between two lobes but not to determine the exact number of viable cells within each lobe. The unknown number of alive cells within BALM and the difficulty to standardize how much proteins is produced by the cells at a certain time point means that instead of an accurate amount of protein produced by BALM, we have to express protein expression as a ratio like ALB/AFP using UPLC-LC/MS/MS. Furthermore, the difficulty to extract alive cells from BALM means techniques such as single-cell RNA sequencing are not directly applicable and requires further development.  Figure 1. Macroscopic appearance of a mouse liver scaffold during the decellularisation process. First, the liver is perfused with EDTA to reduce clotting. Then it is perfused with demineralized water (dH2O) and becomes blanched. After perfusion with sodium deoxycholate (SDC) followed by DNAseI treatment, the liver becomes transparent. Finally, it is washed with dH2O. Scale bar 5 mm.    Figure S5. Immunodetection of different markers on decellularised lobes with no cells. Related to Figures  2, 3 and 4. Non seeded lobes were stained and imaged with the same settings as the seeded lobes for each marker. Some background staining is visible and differences between each marker is due to the different primary antibodies as well as different gain used; however, the diffuse background signal observed is much lower than the primary antibodies signal observed for the seeded lobes. Higher magnification images in the middle and bottom panels correspond to the delineated lower magnification images. Nuclei stained with DAPI. Scale bar 50 µm.

Harvest of organs.
This study was carried out following the recommendations in the Animal (Scientific Procedures) Act 1986. The Home Office approved the study protocol (licence number 70/2716). Organs were obtained from CD-1 mice, any sex, aged 3 to 4 months. Livers were harvested as previously described (Maghsoudlou et al., 2016, Lorvellec et al., 2017. Briefly, immediately following sacrifice by cervical dislocation, the abdominal cavity was opened. The inferior vena cava (IVC) was ligated using a 3/0 silk suture above the right renal vein, whereas the superior vena cava was cut open to allow fluid to flow out during following decellularization. A 24G cannula (Introcan ® -W Certo 24G, Braun, Germany) was inserted to the portal vein (PV) and secured with a 3/0 silk suture (FST, UK), fixing its tip just after the formation of the PV from superior mesenteric and splenic veins. The liver was carefully dissected from surrounding tissues, avoiding injury to the liver capsule, and was transferred to a petri dish filled with 2% EDTA solution. The liver was assessed for injury by injection of EDTA through the cannula, showing sufficient perfusion of each lobe, while also preventing clotting of blood in the microvasculature.
Cells and culture conditions. The hiPSCs line is a generous gift from Prof. L. Vallier (male donor, corrected A1ATD-hiPSCs (Yusa et al., 2011)). This line has successfully been used to generate hepatocyte-like cells in 2D and 3D cultures (Song et al., 2009, Yusa et al., 2011, Lorvellec et al., 2017. Stem cells were cultured in mTeSR Plus medium (Stem cell technologies, UK) on matrigel growth factor reduced (Corning, UK) coated dishes. Media was changed daily or every 2 days and cells were split mechanically every 3-5 days. All cells were tested monthly for the lack of mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, UK). Primary Human Hepatocytes (PHHs, 66 years old male donor) were purchased from Biopredic International (France) as confluent monolayers (ca. 3.08 × 10 5 cells per well) in 24-well plates pre-coated with a single film of collagen. PHHs were maintained in Maintenance media and were transferred to Induction media (Williams E with Glutamax-I added with 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 4 μg/ml of bovine insulin and 5x10 -5 M of hydrocortisone hemisuccinate) for the experiment (Biopredic, France). Human umbilical vein endothelial cells (pooled donors) were purchased from Lonza (UK) and culture in Endothelial Cell Growth Medium 2 (ECGM2, C-22011, Promocell, UK). Cells were expanded for stock generation and frozen at passage 3. HEK293T viral producer cells  were cultured in DMEM high glucose with Glutamax, 10% FBS and 1% penicillin/streptomycin (ThermoFisher Scientific, UK). All cells were cultured in a 5% CO2:95% air humidified atmosphere at 37°C.

Bioreactor.
The bioreactor chamber was composed of a polytetrafluoroethylene body and a polycarbonate lid provided with silicone gaskets and polypropylene connectors. The perfusion system was composed of Pharmed tubings (Cole Parmer, UK), a peristaltic pump (Ismatec Reglo Digital 4-channel 6-rollers; Cole Parmer, UK), and bubble trap (Omnifit Ltd., UK). The air system was composed of a 5% CO2 Air tank connected to flowmeter (Cole Parmer, UK) and Pyrex bottles (Cole Parmer, UK) with caps that allowed sterile tubing connections (Omnifit Ltd., UK). Temperature was controlled using hot plate (Cole Parmer, UK) underneath the bioreactor chamber and the pyrex bottles. pH and temperature monitoring were performed using an invasive sensing system with a sterile dip sensor (Easyferm plus Arc Air 120, Hamilton, UK) directly in contact with the media in the chamber. This sensor is an arc intelligent probe with an integrated transmitter and a Bluetooth connection. It connects to a computer using a wireless adapter (ARC WI 1G Adapter BT; Hamilton, UK) and a wireless converter (Arc Wireless Converter BT; Hamilton, UK).

Decellularisation protocol.
Mouse livers were decellularised as previously described using the Detergent Enzymatic Treatment (DET) method with a few modifications (Maghsoudlou et al., 2016, Lorvellec et al., 2017. Briefly, the PV was connected to a peristaltic pump (Masterflex, UK) perfused with 2% EDTA for 15 min and then with demineralised water (dH2O) (18.2 mΩ/cm) for 24 hours at room temperature followed by 4% sodium deoxycholate (SDC) (Sigma-Aldrich, UK) for 4h. The liver was rinsed overnight with dH2O. The next day, the liver was perfused with 2,000 kU solution of deoxyribonuclease-I (DNase-I, Sigma-Aldrich, UK) in 1 M sodium chloride for 4h. Finally, the liver was perfused with dH2O for 48h. All the reagents were perfused at a rate of 3 ml/min at room temperature. The obtained mouse liver scaffolds were sterilised by gamma-irradiation before seeding cells.

Differentiation of hiHEPs in BALM.
hiPSCs were differentiated into hepatocyte-like cells as described with a few modifications (Sullivan et al., 2010, Lorvellec et al., 2017. Stem cells were cultured until 80-90% confluency. They were split 1/6 on matrigel coated dishes and let rest for 1 day with daily change of mTeSR Plus. They were then incubated with priming media Roswell Park Memorial Institute (RPMI) (Thermofisher, UK) and 1x B27 (ThermoFisher, UK) with 100 ng/ml activin A (Peprotech, UK) and 50 ng/ml Wnt3a (R&D systems, UK) for 3 days followed by 2 days incubation in 100 ng/ml activin A alone. At this point, day 6 of differentiation, Definitive Endoderm-like Cells (DECs) were harvested with enzyme-free Cell Dissociation Buffer (ThermoFisher, UK), avoiding remaining stem cells colonies and seeded in specification medium SR/DMSO (KO/DMEM containing 20% KO Serum Replacement, 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol (ThermoFisher Scientific, UK), and 1% dimethyl sulfoxide (Sigma-Aldrich, UK)) with 10 µM rock inhibitor Y-27632 (Sigma-Aldrich, UK) overnight. DECs, differentiated in parallel, were fixed and analysed afterwards by immunofluorescence staining for Sox17, an endoderm marker, as a low percentage of Sox17 cells at endoderm stage predicts poor hepatocyte differentiation. Differentiation batches that were less than 15% Sox17 positive were excluded from this study. The decellularised mouse liver scaffolds generated by DET were visually evaluated for their ability to be perfused through their vasculature by injecting a coloured media via the catheter in their portal vein. Only the lobes able to be perfused were seeded with DECs. DECs at 2.5-5x10 6 / 200 µl were injected into 3 points of one up to three decellularised lobes with a 29G microfine syringe (BD, UK), this was repeated 3 times drawing up the cells leaking out of the lobe after each injection. A minimum of 5x10 6 cells up to 20x10 6 cells was seeded per scaffold. The seeded decellularised liver was placed into the chamber of the bioreactor with 20 ml of specification media with rock inhibitor and connected to the circuit via its catheter (primed with 10 ml of specification media with rock inhibitor). The flowmeter controlling the gas flow was set at 50 ml/min and the hotplate at 48C so that the temperature of the media inside of the chamber was 37C. The next day, the peristaltic pump was started at a flow rate of 1 ml/min, the media of the chamber was changed to fresh media without rock inhibitor and then every 2 days till day 11 of differentiation. During hepatic maturation 1, growth factors were added every 2 days: 10 ng/ml hepatocyte growth factor HGF and 20 ng/ml oncostatin M (Peprotech, UK), and media was changed every 4 days up to day 17 of differentiation with L-15 medium supplemented with 8.3% FBS, 8.3% tryptose phosphate broth, 10 µM hydrocortisone 21-hemisuccinate, 1 µM insulin (Sigma-Aldrich, UK), 2 mM glutamine, 10 ng/ml HGF and 20 ng/ml oncostatin M. During the hepatic maturation 2, growth factors were added every 2 days: 10 ng/ml HGF and 20 ng/ml oncostatin M, and media was changed every 2 or 4 days up to day 27 of differentiation with William's E medium (Invitrogen, UK) supplemented with 10mM nicotinamide, 17 mM NaHCO3, 0.2 mM AAP2, 20 mM Hepes (Sigma-Aldrich, UK), (Sigma), 6.3 mM NaPyruvate (Invitrogen), 14 mM Glucose (ThermoFisher Scientific, UK), 1x ITS +Premix (Corning, UK), 0.1 µM Dexamethasone (R&D Systems, UK), , 2 mM glutamine, 10 ng/ml HGF and 20 ng/ml oncostatin M. All media after the endoderm stage were supplemented with 100 µg/ml of the antimicrobial Primocin (Invivogen, France).

Coculture of hiHEPs and HUVECs in BALM.
Only the scaffolds able to be perfused were seeded with HUVECs (passages 5-6 maximum), 4 days before seeding with DECs (at day 2 of differentiation). 5x10 6 HUVECs were resuspended into 1 ml of ECGM2 with 10 µM rock inhibitor Y-27632 and slowly perfused with a 1 ml syringe through the PV catheter. The seeded decellularised liver was placed into the chamber of the bioreactor with 20 ml of ECGM2 with rock inhibitor and connected to the circuit via its catheter (primed with 10 ml of ECGM2 with rock inhibitor) overnight. The flowmeter controlling the gas flow was set at 50 ml/min and the hotplate at 48C so that the temperature of the media inside of the chamber is 37C. The next day, the peristaltic pump was started at a flow rate of 0.5 ml/min for half a day and then switched to 1 ml/min. The media of the chamber was changed to fresh media without rock inhibitor and then every 2 days till day 6 of differentiation, when the scaffold was disconnected from the bioreactor and seeded with DECs as described in the previous paragraph. When the scaffold co-seeded with HUVECs and DECs was placed back in the bioreactor, the peristaltic pump was started at a flow rate of 0.5 ml/min for half a day and then switched to 1ml/min. All media were then composed of half media needed for DECs and half ECGM2 till harvesting at day 20-21 of differentiation.

Presto blue cell viability assay.
PrestoBlue™ Cell Viability Reagent (Invitrogen, UK) is a resazurin-based membrane permeable solution, which, upon reduction, forms a red fluorescent compound called resorufin via mitochondrial enzymes of viable cells in the tested systems. As a consequence, the reagent exhibits a change in colour, as well as a shift in its fluorescence. To perform the test the liver scaffolds were disconnected from the chamber of the bioreactor, and dissected into individual lobes in sterile conditions. Subsequently each lobe was cut and incubated in 500 μl of media with 50 μLof Presto Blue reagent in a 24 well dish for 4 h at 37ºC, 5% CO2. 100 μl of media for each lobe was collected and the fluorescence was measured in a 96w plate at λex 544 nm and λem 620 nm using an Envision Multimode Reader and Wallac EnVision software (Perkin Elmer, UK).