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 human liver scaffold comprising (i) providing human liver tissue, (ii) mechanically damaging the cells in the tissue, (iii) subjecting the cells in the tissue to osmotic stress, (iv) optionally exposing the tissue to a protease and/or DNAase, and (v) exposing the tissue to a detergent, and (vi) repeating each of step (iii), step (iv) and step (v) one or more times in any order, thereby producing a human liver scaffold.

2. A method according to claim 1 wherein step (ii) is repeated one or more times.

3. A method according to claim 1 or claim 2 wherein the liver is subjected to multiple cycles comprising one, two or all three of steps (iii), (iv) and (v).

4. A method according to any one of claims 1 to 3 wherein the liver is subjected to multiple cycles comprising step (ii).

5. A method according to any one of the preceding claims wherein the liver tissue is a normal liver tissue.

6. A method according to any one of claims 1 to 4 wherein the liver tissue is pathological liver tissue.

7. A method according to claim 6 wherein the pathological liver tissue displays pathology associated with acute or chronic liver disease.

8. A method according to any one of the preceding claims wherein the cells are mechanically damaging by subjecting the tissue to one or more rounds of freezing and thawing.

9. A method according to any one of the preceding claims wherein the cells are mechanically damaging by subjecting the tissue to HIFU or sonication.

10. A method according to any one of the preceding claims wherein the liver tissue is subjected to osmotic stress by exposing the tissue to a hypotonic reagent.

11. A method according to claim 10 wherein the hypotonic agent is deionised water.

12. A method according to any one of the preceding claims wherein the liver tissue is subjected to osmotic stress by exposing the tissue to a hypertonic reagent.

13. A method according to claim 12 wherein the hypertonic agent is water or saline.

14. A method according to any one of the preceding claims wherein the method comprises step (iv).

15. A method according to claim 14 wherein the tissue is exposed to a protease in step (iv).

16. A method according to claim 15 wherein the protease is trypsin or pronase.

17. A method according to any one of claims 14 to 16 wherein the tissue is exposed to a DNAse in step (iv).

18. A method according to any one of claims 1 to 13 wherein the method does not comprise step (iv).

19. A method according to any one of the preceding claims wherein the detergent is an anionic detergent.

20. A method according to claim 19 wherein the anionic detergent is sodium dodecyl phosphate (SDS) or sodium deoxycholate (SdC).

21. A method according to any one of the preceding claims wherein the detergent is an ionic detergent.

22. A method according to claim 21 wherein the ionic detergent is polyethylene glycol p-(1, 1, 3, 3-tetramethylbutyl)-phenyl ether (Triton X100)

23. A method according to any one of the preceding claims wherein the human liver tissue is exposed to both an ionic detergent and an anionic detergent.

24. A method according to any one of the preceding claims wherein the liver tissue is subjected to flow shear stress during steps (iii) to (vi).

25. A method according to claim 24 wherein the flow shear stress is generated by perfusion.

26. A method according to claim 25 wherein the liver tissue is perfused in a retrograde direction.

27. A method according to claim 25 or claim 26 wherein the perfusion rate is increased to a target value.

28. A method according to claim 27 wherein the initial perfusion rate is 0.1-1.99 ml/min/gram of tissue.

29. A method according to claim 27 or claim 28 wherein the target perfusion rate is 2-20 ml/min/gram of tissue.

30. A method according to any one of claims 25 to 29 wherein the liver tissue is perfused with each decellularisation reagent for about 0.004 to about 4 hours per gram of solid tissue.

31. A method according to any one of claims 25 to 30 wherein step (vi) comprises repeating steps (iii) and (v) 1 to 25 times by perfusion through the liver tissue.

32. A method according to any one of claims 25 to 31 wherein step (vi) comprises repeating steps (iii) to (v) in the following sequence by perfusion through the liver tissue; (iii), (iv), (iii), (iv) (v), [(iii), (v)].sub.n, (iii), (iv), [(iii), (v)].sub.n, where n is 1 to 25.

33. A method according to any one of claims 25 to 31 wherein step (vi) comprises repeating steps (iii) and (v) 1 to 25 times by perfusion through the liver tissue.

34. A method according any one of claims 31 to 33 wherein step (ii) is repeated one or more times.

35. A method according to any one of claims 25 to 34 wherein the liver tissue is subjected to a perfusion regime as set out in Table 1.

36. A method according to any one of claims 25 to 35 wherein the liver tissue is a whole liver or a functional unit thereof.

37. A method according to any one of claims 1 to 24 wherein the flow shear stress is generated by agitation.

38. A method according to claim 37 wherein the liver tissue is immersed in the decellularisation reagents and agitated at 100-1000 rpm.

39. A method according to claim 37 or claim 38 wherein the liver tissue is a non-vascularized liver section of 2 cm.sup.3 or less.

40. A method according to any one of claims 37 to 39 wherein the agitated tissue is subjected to one or more cycles of; (a) subjecting the tissue to osmotic stress (b) exposing the tissue to a protease and/or DNAase, and (c) exposing the tissue to a detergent, wherein (a), (b) and (c) may occur in any order in each cycle.

41. A method according to claim 40 wherein the tissue is exposed to the hypotonic reagent for 12 to 36 hours.

42. A method according to any one of claims 37 to 41 wherein the tissue is exposed to the detergent for 12 to 36 hours.

43. A method according to any one of claims 37 to 42 wherein the tissue is exposed to the DNAse for about 3 hours.

44. A method according to any one of claims 37 to 43 wherein the agitated tissue is subjected to one or more cycles of; (a) subjecting the tissue to osmotic stress, (b) exposing the tissue to a protease, (c) exposing the tissue to an anionic detergent, (d) exposing the tissue to an ionic detergent.

45. A method according to claim 43 wherein the tissue is subjected to osmotic stress for 15 mins to 3 hours.

46. A method according to claims 37 to 45 wherein the tissue is exposed to the protease for 12 to 36 hours at ambient temperature.

47. A method according to any one of claims 37 to 46 wherein the tissue is exposed to the anionic detergent for 12 to 96 hours.

48. A method according to any one of claims 37 to 47 wherein the tissue is exposed to the ionic detergent for 12 to 72 hours.

49. A method according to any one of claims 37 to 48 wherein the duration of each cycle of exposure to the decellularisation reagents is 1-3 days.

50. A method according to any one of claims 37 to 49 wherein the wherein the liver tissue is subjected to a regime as set out in any one of Tables 3 to 6.

51. A method according to any one of claims 1 to 50 comprising sterilising the scaffold following decellularisation.

52. A method according to any one of claims 1 to 51 comprising re-populating the scaffold with cells to produce artificial liver tissue.

53. A method according to claim 52 wherein the cells are human primary and cell line liver cells, human primary hepatocytes, endothelial cells, human iPSCs or cells derived from patient-specific iPSCs, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSC), human fetal stem cells, human cancer cells and human endothelial progenitor cells (EPCs).

54. A human liver scaffold or artificial liver tissue produced by a method according to any one of claims 1 to 53.

55. Use of a human liver scaffold or artificial liver tissue for in vitro disease modelling.

56. A method of disease modelling comprising; providing a human liver scaffold or artificial human liver tissue produced by a method according to any one of claims 1 to 53, and, determining the effect of a compound, drug, biological agent, device or therapeutic intervention on the scaffold or tissue.

57. A method of diagnosing liver disease in a human individual may comprise; providing a sample of liver scaffold from the individual produced by a method according to any one of claims 1 to 53, determining the presence and amount of one or more liver scaffold proteins in the sample.

58. A method of treatment of a liver disease or dysfunction comprising; implanting a human liver scaffold or artificial human liver tissue produced by a method according to any one of claims 1 to 53 into an individual in need thereof.

59. A human liver scaffold or artificial human liver tissue produced by a method according to any one of claims 1 to 53 for use in the treatment of liver disease or dysfunction in an individual.

60. A method of treatment of a liver disease or dysfunction comprising; connecting an extra corporeal human artificial human liver tissue produced by a method according to claim 52 or 53 to the vascular system of an individual in need thereof, such that circulating blood from the individual passes through the tissue and is returned to the individual.

61. A bioreactor comprising; a human liver scaffold produced by a method according to any one of claims 1 to 51; an input conduit for the introduction of culture medium to the scaffold; and an output conduit for the exit of culture medium from the scaffold.

62. A bioreactor according to claim 61 wherein the human liver scaffold is seeded with cells.

63. A bioreactor comprising; an artificial human liver tissue produced by a method according to claim 52 or claim 53; an input conduit for the introduction of blood to the scaffold or artificial human liver tissue; and an output conduit for the exit of blood from the scaffold or the artificial human liver tissue.

Description

[0214] FIG. 1 shows flow rate (in ml/min) relative to the different phases of perfusion over the 14 days of the decellularisation process.

[0215] FIG. 2 shows 125=.sup.3 Liver Tissue Cubes (LTCs) before decellularisation.

[0216] 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).

[0217] 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).

[0218] 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.

[0219] 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).

[0220] 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.

[0221] FIG. 8 shows FACS staining of SK-Hep with anti-human Nanog (B) and Isotype control (A).

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] FIG. 15 shows a low magnification (90) SEM image including a portal tract surrounded by a typical lobular structure.

[0229] FIG. 16 shows a 300 SEM image confirming scaffold acellularity and clearly defining spaces once occupied by hepatocytes (i.e. hepatocyte-free spaces).

[0230] 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.

[0231] 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.

1. Methods

1.1 Human Liver Harvest and Cannulation

[0232] 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 24h hours to facilitate cell lysis.

1.2 Perfusion Decellularisation of the Whole Human Liver Left Lobe

[0233] 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.

[0234] 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.

1.3 Agitation Decellularisation of Human LTC

[0235] The LTCs were thawed at 37 C for 1-1.5h. The protocol for the decellularisation of LTCs is shown in Tables 3 to 5.

2. Results

[0236] 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).

[0237] 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).

[0238] 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).

[0239] 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).

[0240] 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).

[0241] 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).

[0242] 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).

[0243] 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).

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

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

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

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

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

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