Source of human livers

The work described in this paper was performed on healthy human livers that were harvested for transplantation and then judged unsuitable because of prolonged graft cold ischaemic time, the presence of extra-hepatic malignancy or other important extra-hepatic co-morbidities in donors or recipients. Livers included in this study were defined “healthy” because of the absence of any degree of tissue fibrosis and fat accumulation by histological analysis. The study was approved by the UCL Royal Free BiobankEthical Review Committee (NRES Rec Reference: 11/WA/0077). Informed consent for research was confirmed via the NHSBT ODT organ retrieval pathway and the project was also approved by the NHSBT Research Governance Committee. Donor livers were processed in accordance with the UCL Royal Free Biobank protocols under the Research Tissue Bank Human Tissue Act licence, prior to use in research. Human livers obtained at the Royal Free London Foundation Trust were coordinated, received and recorded by the UCL Tissue Access for Patient Benefit organisation (TAPb) which links research activities between UCL Royal Free Biobank, the Royal Free Trust and UCL. TAPb has full governance in place for this purpose which has involved NHSBT ODT pathway, the Human Tissue Authority licencing and the local Trust/UCL Research offices.

Surgery of human liver in preparation for the decellularization procedure

Human livers (n = 3) were either surgically processed in order to obtain the liver left lobes (n = 2) [374 g and 250 g, respectively] including segments 1, 2, 3 and 4 or retained as a whole liver (n = 1) [1, 774 Kg] with preserved vascular access.

Whole liver preparation: the donor liver was processed exactly as for liver transplantation; thereafter the upper caval cuff, reaching the atrial rim, was oversewn with a running double row of 3-0 prolene suture. The water tightness of the organ was proofed both by antegrade portal and retrograde hepatic venous perfusion under relative high pressure as produced by a 50 ml bladder syringe with PBS solution.

Segmental liver preparation of left lateral liver (S1 + S2 + S3 + S4): dissection is started at the hilar region with inspection, preparation, division and ligation of the hepatic artery (HA), of the portal vein (PV) and of the bile duct (BD). Segment 4 HA is preserved if originating from the right hepatic artery, forcing the division of the right HA distal to its bifurcation. The right hepatic vein is divided and the middle and left hepatic veins are preserved with the future left reduced size left liver. The line of parenchymal division is maintained to the left of the line of Cantlie in order to maintain the left portion of S4. The parenchyma is divided sharply with a knife for the liver capsule and subsequently with crush-clamp technique, performing meticulous hemo- and bilio-stasis. The parenchymal division is carried to the end, producing a left lateral liver extended to S4 and a right liver as non-functional unit. Finally, the upper caval cuff, reaching the atrial rim is oversewn with a running double run of 3-0 prolene suture.

Human livers were then frozen at −80 °C for at least 24 h for the purposes of initial destruction of the various cellular compartments.

Decellularization Protocols of Whole Human Liver Lobe

The perfusion regime for the decellularization of the liver left lobe is shown in Table1. The decellularization of the whole liver was achieved by repeating the procedure shown in Table 1 three times (the freezing/thawing step was performed just once). The following abbreviations were used: distilled Water (dH 2 O), TX100 (Triton X100), SDS (sodium dodecyl sulfate), A-A 5% (Antibiotic and Antimycotic), PAA (paracetic acid) and EtOH (ethanol) purchased from Sigma Aldrich.

Table 1 Perfusion protocol. Full size table

The initial flow rate for decellularization perfusion was 0.2–0.3 ml/min/g of liver. Subsequently, two phases of perfusion were adopted a) steeply increasing flow rate to compensate reduced resistance and b) stabilization of the flow rate as the decellularization proceeds. The two phases of flow rate are shown in Fig. 8. After decellularization, 125 mm3 cubic scaffold fragments (5 mm×5 mm×5 mm) were dissected by scalpel cleavage for further characterization and liver bioengineering in vitro.

Figure 8 Flow rate. The graph illustrates the increasing flow rate (x fold) over time starting with an initial flow rate of 0.2–0.3 ml/min/g of liver. The decellularization of a human liver left lobe (blue line) was achieved in 2 weeks, while the decellularization of a whole human liver (red line) was completed in 6 weeks. Full size image

Histology and immunostaining analysis

Samples were fixed for at least 24 hours in 10% neutral buffered formalin solution (pH 7.4) at RT. Tissue was embedded in paraffin and sectioned at 4 μm. Prior to staining sections were dewaxed in xylene and rehydrated using graded industrial denatured alcohol (IDA).

Histochemical stains: tissue sections were stained with Harris’s Haematoxylin and Eosin (H&E) (Leica, Germany), Picro-Sirius Red (SR) (Hopkin & Williams) (BDH Chemicals Ltd, Cellpath Ltd) and stains Miller’s Elastic stain with a Picro-Sirius red counter stain (VWR, Leica, Raymond A Lamb).

Immunocytochemistry: sections stained with Collagen I, III, IV, fibronectin and laminin were incubated in 0.5% Trypsin (MP Biomedical)/0.5% Chymotrypsin (Sigma)/1% Calcium Chloride (BDH) in Tris buffered saline pH 7.6 (TBS) for 30 minutes at 37 °C. Sections stained with alpha-smooth muscle actin (SMA) were microwaved (640 W) for 20 minutes in 1L of Tris-EDTA buffer (10 mM Tris-base/1 mM EDTA solution, pH9.0) and sections for CD3 pressure cooked for 3 mins in sodium citrate buffer (10 mM Sodium Citrate, pH 6.0). Slides were then soaked in TBS with 0.04% Tween-20 (Sigma) for 5 mins, blocked in peroxidase blocking solution (Novocastra) for 5 minutes, washed in TBS for 5 mins and then incubated for 1 hour in the following primary antibodies; collagen I (Rabbit pAb to coll1 (ab34710), diluted 1:200; Abcam), collagen III (Rabbit pAB to coll3 (ab7778), diluted 1:500; Abcam), collagen IV (mouse mAb to coll4 (M0785), diluted 1:25; Dako), fibronectin (mouse mAb to fibronectin (MAB1937), diluted 1:100; Millipore), laminin (mouse mAb to laminin α5-chain (MAB1924), diluted 1:200; Millipore), alpha-Smooth Muscle Actin (mouse mAb to SMA, (M0851/1A4), diluted 1:500; Dako) and CD3 (rabbit pAb to CD3, (AO452), diluted 1:200; Dako). The slides were then placed for 25 minutes in NovolinkTM post primary (Novocastra), 25 mins in NovolinkTM polymer solution (Novocastra) and developed with NovolinkTM 3,3′ di-amino-benzidine (Novocastra) with a 5 minute wash in TBS with 0.04% Tween-20 between each step. Slides were counterstained with Mayer’s Haematoxylin (Sigma) for 3 min. All sections were dehydrated in graded IDA, cleared in xylene and were mounted with DPX (Leica biosystems); cover slipped and observed using a Zeiss Axioskop 40. Images were captured with an Axiocam IcC5 using Zeiss Axiovision (verison 4.8.2). All images were analysed and enhanced using Fiji v1.49d (ImageJ Jenkins server).

DNA quantification

To assess total DNA content within native tissue and acellular matrices, the DNeasy Blood and Tissue kit was used according to the manufacturer’s manual (Qiagen). Briefly, specimens were digested with Proteinase K overnight. DNA samples were purified using buffers provided by the company and measured spectrophotometrically (Nanodrop, Thermo Scientific, US). Optical densities at 260 nm and 280 nm were used to estimate the purity and yield of nucleic acids.

Collagen quantification

The collagen content of native tissue and decellularized tissue was quantified using the total collagen assay kit according to the manufacturer’s manual (QuickZyme Biosciences, The Netherlands). Briefly, samples were hydrolysed in 6M HCl at 95 °C for 20 hours, the hydrolysates were mixed with a chromogen solution staining the hydroxyproline residues and color was developed at 60 °C for 1 hour. The absorbance for each sample was determined at 555 nm using a FLUOstar Omega microplate reader (BMG labtech, Germany) and the collagen quantity was calculated by usage of a standard curve of pure collagen hydrolysates.

Elastin quantification

The elastin content of native and decellularized tissue was quantified using the FASTIN elastin assay (Biocolor, UK) according to the manufacturer’s instructions. Briefly, the samples were homogenized and elastin was solubilized in 0.25 M oxalic acid. Two consecutive incubations were performed at 95 °C to ensure complete extraction of elastin. Extracts were incubated with 5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonate (TPPS) dye and absorbance was determined at 513 nm spectrophotometrically FLUOstar Omega microplate reader (BMG labtech, Germany). Elastin concentrations from a standard curve were used to calculate the elastin content of the tissue.

Scanning Electron Microscopy (SEM)

Samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and left for 24 hours at 4 °C. Following washing with 0.1 M phosphate buffer, samples were cut into segments of approximately 1 cm length and cryoprotected in 25% sucrose, 10% glycerol in 0.05 M PBS (pH 7.4) for 2 hours, then fast frozen in Nitrogen slush and fractured at approximately −160 °C. Next, samples were then placed back into the cryoprotectant at room temperature and allowed to thaw. After washing in 0.1 M phosphate buffer (pH 7.4), the material was fixed in 1% OsO4 / 0.1 M phosphate buffer (pH 7.3) at 3 °C for 1½ hours and washed again in 0.1 M phosphate buffer (pH 7.4). After rinsing with dH2O, specimens were dehydrated in a graded ethanol-water series to 100% ethanol, critical point dried using CO 2 and finally mounted on aluminum stubs using sticky carbon taps. The fractured material was mounted to present fractured surfaces across the parenchyma to the beam and coated with a thin layer of Au/Pd (approximately 2 nm thick) using a Gatan ion beam coater. Images were recorded with a 7401 FEG scanning electron microscope (Jeol, USA).

Xenotransplantation in immunocompetent mice

All animal experiments were approved by the Home Office under the UK Animals and Scientific Procedures Act 1986 and in accordance with the guidelines of the Comparative Biology Unit, Biological Services University College London (UCL) under Project license 70/7100.

For biocompatibility studies, twelve male C57BL/6J mice, aged 3–4 weeks, were used. Human liver cubic scaffolds were surgically implanted either subcutaneously (n = 6) or into the omentum (n = 6). Following shaving the operating area, the skin was cleaned with 10% povidone iodine (Videne, Ecolab, Leeds, UK) and 20% chlorhexidine gluconate (Hydrex pink, Ecolab, Leeds, UK). Isoflurane (2% with 98% oxygen) was used to induce anesthesia. For subcutaneous implantation, a small incision (5 mm) was made between the shoulder blades and a subcutaneous tunnel was made by blunt dissection. The human liver cubic scaffold was inserted into the subcutaneous tunnel and the wound was closed with absorbable 4-0 vicryl sutures. When applying an omental implantation, a small midline abdominal incision was made and the human liver cubic scaffold was folded with the omentum and was secured in place by 6-0 vicryl sutures. The abdomen was closed in two layers. After 7 and 21 days, mice were euthanized and the implants along with the surrounding tissues were harvested and fixed in 10% formalin for histological and immunohistochemical evaluation.

Cell Culture

The LX2 cell line is a well-established hepatic stellate cell line that was generated by a spontaneous immortalization in low serum conditions30. Cells are cultured in Iscove’s Modified DMEM supplemented with 2 mM/L glutamine, 0.1 mM/L non-essential amino acids, 1.0 mM/L sodium pyruvate and 20% Foetal Bovine Serum (FBS). HepG2 and Sk-Hep-1 cells (ATCC® HTB-52™) are derived from a human hepatoblastoma and a human hepatocellular carcinoma, respectively. Both cell types were purchased from ATCC (VA, USA) and cultured in Eagle’s Minimum Essential medium (EMEM), supplemented with Glutamax, 0.1 mM/L non-essential amino acids, 1.0 mM/L sodium pyruvate and 10% FBS. All cells were cultured under standard conditions in a humidified incubator under 5% CO 2 and at 37 °C. Every 3 days the complete culture medium was changed and sub-confluent cells were trypsinized and passaged at a split ratio 1:3.

Repopulation and culture of engineered human liver

Human liver cubic scaffolds were kept overnight in complete medium [day -1]. Cells were re-suspended at a concentration of 2 million cells per 50 μl (2 × 106/50 μL) per scaffold (n ≥ 12 per cell line). Cells were drawn up in a 0.5 ml insulin syringe and released drop by drop to finally cover the decellularized tissue. Seeded scaffolds were kept for 2 h in a humidified environment at 37 °C with 5% CO 2 allowing cell attachment followed by addition of complete culture medium [day 0]. The culture medium was changed at day 1 and afterwards every 3 days. At days 7, 14 and 21 following seeding, the scaffolds were placed in 10% formaldehyde and assessed by histology and immunohistochemistry or fixed in 2.5% glutaraldehyde for SEM analysis.

Statistical analysis

Results were expressed as mean ± s.d. All data was analysed with ANOVA or Student’s t-test. Two-talied p values less than 0.05 were considered statistically significant.