Human liver scaffolds

Abstract

This invention relates to methods for decellularising human liver tissue to produce human hepatic extracellular matrix (ECM) scaffolds, for example for use in therapy or disease modelling. The methods involve mechanically damaging cells in the tissue, for example by freeze thaw, and then subjecting the liver tissue to multiple cycles of osmotic stress, detergent treatment and protease and/or DNAase treatment to produce a decellularised human ECM scaffold.

Claims

1. A method of producing a decellularized human liver scaffold comprising (i) providing healthy or pathological human liver tissue comprising a whole liver or a functional unit thereof, (ii) mechanically damaging the cells in the tissue, (iii) subjecting the cells in the tissue to osmotic stress by exposing the tissue to a hypotonic reagent or hypertonic reagent, (iv) exposing the tissue to a protease and/or DNAase, and (v) exposing the tissue to a detergent, and (vi) repeating steps (iii) to (v) in the following sequence by perfusion through the liver tissue: (iii), (iv), (iii), (iv), (v), [(iii), (v)].sub.n, optionally [(iii), or (iv)], [(iii), (v)].sub.n, where n is independently 1 to 25; thereby producing a decellularized human liver scaffold, wherein the human liver tissue is subjected to flow shear stress generated by perfusing the human liver tissue in a retrograde direction with the hypotonic reagent, hypertonic reagent, protease and/or DNAase, and detergent during steps (iii) to (vi) at a perfusion rate which increases from an initial value of 0.1-1.99 ml/min/gram of tissue and stabilizes to a target value of 2-20 ml/min/gram of tissue.

2. A method according to claim 1 wherein the cells are mechanically damaged by subjecting the tissue to one or more rounds of freezing and thawing, or by subjecting the tissue to high intensity focused ultrasound (HIFU) or sonication.

3. A method according to claim 1 wherein the hypotonic agent is deionised water.

4. A method according to claim 1 wherein the tissue is exposed to a trypsin or pronase in step (iv).

5. A method according to claim 1 wherein the detergent is an anionic detergent.

6. A method according to claim 5 wherein the anionic detergent is sodium dodecyl sulfate (SDS) or sodium deoxycholate (SdC).

7. A method according to claim 1, wherein the detergent is a non-ionic detergent, and is polyethylene glycol p-(1, 1, 3, 3-tetramethylbutyl)-phenyl ether (Triton X100).

8. A method according to claim 1 wherein the liver tissue is subjected to a perfusion regime as set forth below: TABLE-US-00007 DAY Steps and Reagents DAY 1 Thaw the liver o.n. at 4 C. DAY 0 dH2O 0.025% Trypsin/EDTA DAY 1 dH2O 0.01% SDS 0.1% SDS 1% SDS DAY 2 dH2O 0.025% Trypsin/EDTA 1% SDS DAY 3 dH2O 3% TX100 DAY 4 dH2O 3% TX100 DAY 5 dH2O 3% TX100 DAY 6 dH2O 3% TX100 DAY 7 dH2O 0.025% Trypsin/EDTA DAY 8 dH2O 1% SDS DAY 9 dH2O 1% SDS DAY 10 dH2O 1% SDS DAY 11 dH2O 1% SDS DAY 12 dH2O 1% SDS DAY 13 dH2O PBS/Antib-Antimic 5% 3% TX100 DAY 14 dH2O PBS PBS/Antib-Antimic 5% dH2O 0.1% PAA/4% EtOH PBS.

9. A method according to claim 1 comprising sterilising the scaffold following decellularisation.

10. A method according to claim 1 comprising re-populating the decellularized human liver scaffold with cells to produce artificial liver tissue.

11. A method according to claim 10 wherein the cells are selected from the group consisting of human primary and cell line liver cells, human primary hepatocytes, human endothelial cells, human induced pluripotent stem cells (iPSCs) or cells derived from patient-specific iPSCs, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs), human fetal stem cells, human cancer cells and human endothelial progenitor cells (EPCs).

12. A method according to claim 1, further comprising: providing a sample of the decellularized human liver scaffold, wherein the human liver tissue was obtained from a healthy or pathological human liver, and determining the presence and amount of one or more liver scaffold proteins in the sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

(2) FIG. 1 shows flow rate (in ml/min) relative to the different phases of perfusion over the 14 days of the decellularisation process.

(3) FIG. 2 shows 125=.sup.3 Liver Tissue Cubes (LTCs) before decellularisation.

(4) FIG. 3 shows histological comparison of native tissue and decellularised LTCs (dLTCs) after 4 decellularisation cycles. H&E (Haematoxylin and Eosin) staining showed removal of cells after LTCs decellularisation and SR (Sirius Red) staining showed preservation of collagen (red) and removal of cellular materials (yellow).

(5) FIG. 4 shows quantitative measurement of Collagen (A) and DNA (B) after decellularisation. Collagen quantification following different agitation speeds (900C1-900C4) demonstrated a preservation of the amount of collagen when compared to fresh tissue. Decellularisation was efficient with a marked decrease in DNA content (p<0.01) already after 1 treatment cycle (B).

(6) FIG. 5 shows repopulation of human liver scaffolds with the human hepatic stellate cell line LX2. (A) H&E staining demonstrates progressive LX2 cell migration into the LTC scaffold when comparing day 1 with 21 days after recellularisation. (B) The process of repopulation is characterized by marked cell proliferation as detected with an immunostaining for the proliferation marker Ki67.

(7) FIG. 6 shows an increase in the total cell count after 14 days of repopulation with LX2. Importantly, the total cell count in human liver scaffolds significantly increased between 14 and 21 days (A). KI67 immunohistochemistry shows that more than 85% of the cells were proliferating at all different time points (B).

(8) FIG. 7 shows repopulation of decellularised human liver scaffolds with the human hepatocellular carcinoma cell line SK-Hep. H&E and HVG (Haematoxylin Van Gieson) staining demonstrates cell attachment after 1 day of bioengineering and progressive cell migration into the human liver scaffold when comparing day 1 with day 14 after recellularisation.

(9) FIG. 8 shows FACS staining of SK-Hep with anti-human Nanog (B) and Isotype control (A).

(10) FIG. 9 shows repopulation of decellularised human liver scaffolds with human hepatocellular carcinoma cell line SK-Hep after 1 and 7 days. Nanog expression was analysed by immunohistochemistry. It is evident that the cells attached to the ECM at day 1 and the cells which migrating within the scaffold at day 7 are Nanog positive cells.

(11) FIG. 10 shows repopulation of decellularised human liver scaffolds with human hepatocellular carcinoma cell line SK-Hep after 14 days. Nanog expression was analysed by immunohistochemistry (Brown) and cells were counterstained with Hematoxilin (Blue). The process of repopulation after 14 days is characterized by both Nanog positive and Nanog negative cells (B). It is remarkable that at this stage Nanog positive cells surround the repopulated scaffold (A) with features resembling the development of human hepatocellular carcinoma.

(12) FIG. 11 shows the macroscopic appearance of the decellularised human liver left lobe using two different light backgrounds to highlight the preservation of the vascular tree.

(13) FIG. 12 shows histological sections of the decellularised vascular (portal vein, hepatic artery and hilar plate) and biliar tree. H&E shows absence of cells in the decellularised tissues. SR and EVG stainings show collagen (red) and elastin (blue) preservation, respectively.

(14) FIG. 13 shows histological comparison of fresh liver tissue and the decellularised human liver left lobe segments (S1, S2, S3 and S4). H&E staining showed removal of cells after decellularisation and SR and EVG (Elastic Van Gieson) stainings show collagen (red) and elastin (blue) preservation, respectively.

(15) FIG. 14 shows repopulation of human liver scaffolds with the human hepatic stellate cell line LX2. H&E and HVG stainings demonstrate cell attachment after 1 day of bioengineering and progressive cell migration into the human liver scaffold when comparing day 1 with day 14 after recellularisation.

(16) FIG. 15 shows a low magnification (90) SEM image including a portal tract surrounded by a typical lobular structure.

(17) FIG. 16 shows a 300SEM image confirming scaffold acellularity and clearly defining spaces once occupied by hepatocytes (i.e. hepatocyte-free spaces).

(18) FIG. 17 shows a high magnification (2500) SEM image demonstrating an exceptionally preserved three-dimensional meshwork of connective tissue fibres structuring the hepatocyte-free spaces.

(19) Abbreviations; SdC Sodium Deoxycholate; PBS/AA PBS+Antibiotic Antimycotic; T/E 0.025% Trypsin/EDTA 0.025%; SDS Sodium Dodecyl Sulfate; TX100 Triton X 100; RT Room Temperature; PAA Paracetic Acid; EtOH Ethanol.

(20) 1. Methods

(21) 1.1 Human Liver Harvest and Cannulation

(22) Discarded Human Liver Organs (DHLO) unsuitable for liver transplantation were heparinized according to the standard procedure for transplantation. DHLOs are adequately prepared by multiple block subdivision in small non-vascularized liver units of 0.2-1.0 cm (Liver Tissue Cubes, LTC) and/or by segmental or sub-segmental preparation of units provided of vascular-biliary pedicles. The whole human liver or alternatively the left lobe (segments 2-3-41), right lobe (segments 5-8) or the left lateral liver (Segments 2-31) as well as the Liver Tissue Cubes (LTC) were frozen at 80 C for at least 24 h hours to facilitate cell lysis.

(23) 1.2 Perfusion Decellularisation of the Whole Human Liver Left Lobe

(24) First, the whole human liver lobe was thawed overnight in PBS at 4 C. Secondly, the vena cava or the hepatic veins were cannulated to initiate a retrograde perfusion system.

(25) Finally, the 5CDFs were applied to achieve full organ decellularisation, as shown in Table 1, 2 and FIG. 1. Two phases of perfusion were adopted a) steeply increasing flow rate to compensate resistance b) stabilization of the flow rate as the decellularisation proceeds. The two phases of flow rate are shown in FIG. 1.

(26) 1.3 Agitation Decellularisation of Human LTC

(27) The LTCs were thawed at 37 C for 1-1.5 h. The protocol for the decellularisation of LTCs is shown in Tables 3 to 5.

(28) 2. Results

(29) Native liver tissue and decellularised LTCs (dLTCs) were compared histologically after 4 decellularisation cycles. The decellularisation cycles were found to have removed cells and cellular material from the LTCs, whilst preserving collagen (FIG. 3).

(30) Collagen and DNA were quantified in the dLTCs after decellularisation. The amount of collagen in the dLTCs was found to be preserved at different agitation speeds when compared to fresh tissue (900C1-900C4) (FIG. 4A). Decellularisation was efficient with a marked decrease in DNA content (p<0.01) after only 1 treatment cycle (FIG. 4B).

(31) Decellularised human LTCs were repopulated with the human hepatic stellate cell line LX2. The LX2 cells were found to progressively migrate into the LTC scaffold over 21 days after recellularisation (FIG. 5A). The total cell count in the human liver scaffolds was found to increase after 14 days of repopulation with LX2 and significantly increased between 14 and 21 days (FIG. 6A). Immunostaining for the proliferation marker Ki67 showed that the process of repopulation is characterized by marked cell proliferation (FIG. 5B) and at all different time points, more than 85% of the cells were proliferating (FIG. 6B).

(32) Decellularised human liver scaffolds were repopulated with the human hepatocellular carcinoma cell line SK-Hep. Cell attachment was observed after 1 day of bioengineering and the SK-Hep cells progressively migrated into the human liver scaffold in the 14 days after recellularisation (FIG. 7). Cells attached to the ECM at day 1 and the cells which migrating within the scaffold at day 7 were shown to be Nanog positive cells (FIGS. 8 and 9). The process of repopulation after 14 days was characterized by both Nanog positive and Nanog negative cells (FIG. 10). Remarkably, after 14 days, Nanog positive cells were found to surround the repopulated scaffold with features resembling the development of human hepatocellular carcinoma (FIG. 10A).

(33) The vascular tree of the human liver left lobe was found to be preserved observed following decellularisation (FIG. 11). Cells were found to be absent from the decellularised tissues whilst collagen and elastin were preserved (FIG. 12).

(34) Comparison of fresh liver tissue and the decellularised human liver left lobe segments (S1, S2, S3 and S4) confirmed the removal of cells after decellularisation and the preservation of collagen and elastin (FIG. 13).

(35) Decellularised human liver left lobe segments were repopulated with the human hepatic stellate cell line LX2. The LX1 cells were found to attach to the decellularised scaffold after 1 day and to progressively migration into the scaffold over 14 days (FIG. 14).

(36) Decellularised human liver scaffolds were analysed by scanning electron microscopy. The SEM images confirmed scaffold acellularity and showed the presence of clearly defined spaces once occupied by hepatocytes (i.e. hepatocyte-free spaces). The three-dimensional meshwork of connective tissue fibres structuring the hepatocyte-free spaces, as well as portal tracts and lobular structure, were found to be an exceptionally preserved (FIGS. 15-17).

(37) TABLE-US-00001 TABLE 1 DAY Steps and Reagents DAY 1 Thaw the liver o.n. at 4 C. DAY 0 dH2O 0.025% Trypsin/EDTA DAY 1 dH2O 0.01% SDS 0.1% SDS 1% SDS DAY 2 dH2O 0.025% Trypsin/EDTA 1% SDS DAY 3 dH2O 3% TX100 DAY 4 dH2O 3% TX100 DAY 5 dH2O 3% TX100 DAY 6 dH2O 3% TX100 DAY 7 dH2O 0.025% Trypsin/EDTA DAY 8 dH2O 1% SDS DAY 9 dH2O 1% SDS DAY 10 dH2O 1% SDS DAY 11 dH2O 1% SDS DAY 12 dH2O 1% SDS DAY 13 dH2O PBS/Antib-Antimic 5% 3% TX100 DAY 14 dH2O PBS PBS/Antib-Antimic 5% dH2O 0.1% PAA/4% EtOH PBS

(38) TABLE-US-00002 TABLE 2 DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 100 300 425 475 550 675 850 1300 1500 1750 1750 1800 1750 1750 1750 150 350 450 500 600 700 1000 1350 1550 1755 1800 1750 1750 1750 1750 200 375 450 525 650 750 1200 1400 1700 1850 1800 1750 1750 1750 1750 250 400 450 525 650 800 1200 1400 1700 1900 1800 1750 1750 1750 1750 1700 1750

(39) TABLE-US-00003 TABLE 3 Time Reagents Temperature rpm 24 h dH20 4 C. 900 4-6 h SdC 4% RT 900 5 min PBS RT 900 3 h Dnase RT 900 5 min PBS/AA RT 900

(40) TABLE-US-00004 TABLE 4 Time Reagents Temperature rpm 12-36 h dH20 4 C. 100-1000 4-12 h SdC 4% RT 100-1000 5-30 min PBS RT 100-1000 3 h Dnase RT 100-1000 5-30 min PBS/AA RT 100-1000

(41) TABLE-US-00005 TABLE 5 Time Reagents Temperature rpm 15-30 min dH20 RT 100-1000 12-36 h T/E0.025% RT 100-1000 12-72 h SDS 0.01-1% RT 100-1000 12-72 h TX100 3% RT 100-1000 5-30 min PBS/AA RT 100-1000

(42) TABLE-US-00006 TABLE 6 Calculated Overall Agitating Speed G-force Reagents (1.2 ml) Time System (rpm) (g) OS-1 1. Deionised water, 24 hrs 8 days Labnet - 900 0.432 2. PBS 1%, 5 mins 16 days Orbit 3. SDC 4%, 5.5 hrs M60 4. PBS 1%, 5 mins microtube 5. DNase solution, 3 hrs shaker 6. PBS 1%, 5 mins 7. Repeated steps 1-6 a total of 4 and 8 times OS-D 1. Alternate between deionised 8 days Labnet 900 0.432 water and dextrose solution, 1 hr Orbit each for 8 hrs M60 2. Deionisd water, 16 hrs microtube 3. PBS 1%, 5 mins shaker 4. SDC 4%, 5.5 hrs 5. PBS 1%, 5 mins 6. DNase solution, 3 hrs 7. PBS 1%, 5 mins 8. Repeated steps 1-7 a total of 4 times MS-1 1. Deionised water, 24 hrs 8 days Magnetic 300-400 5.0-8.9 2. PBS 1%, 5 mins Stirrer 3. SDC 4%, 5.5 hrs 4. PBS 1%, 5 mins 5. DNase solution, 3 hrs 6. PBS 1%, 5 mins 7. Repeated steps 1-6 a total of 4 times MS-S 1. Deionised water, 24 hrs 8 days Magnetic 300-400 5.0-8.9 2. PBS 1%, 5 mins Stirrer 3. SDC 4%, 5.5 hrs 4. PBS 1%, 5 mins 5. DNase solution, 3 hrs 6. PBS 1%, 5 mins 7. Saline Solution 9%, 14.5 hrs 8. Repeated steps 1-7 a total of 4 times