(19)
(11)EP 3 328 992 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
04.11.2020 Bulletin 2020/45

(21)Application number: 16750213.7

(22)Date of filing:  29.07.2016
(51)International Patent Classification (IPC): 
A61P 1/00(2006.01)
A61P 1/18(2006.01)
A61P 11/00(2006.01)
A61P 13/12(2006.01)
A61P 17/00(2006.01)
A61P 19/04(2006.01)
A61P 21/00(2006.01)
A61P 43/00(2006.01)
C12M 1/33(2006.01)
B01L 3/00(2006.01)
A61P 1/16(2006.01)
A61P 9/00(2006.01)
A61P 13/08(2006.01)
A61P 15/00(2006.01)
A61P 19/02(2006.01)
A61P 19/08(2006.01)
A61P 27/02(2006.01)
C12N 5/071(2010.01)
A61L 27/36(2006.01)
A61P 13/10(2006.01)
(86)International application number:
PCT/GB2016/052349
(87)International publication number:
WO 2017/017474 (02.02.2017 Gazette  2017/05)

(54)

METHODS FOR THE PRODUCTION OF DECELLULARISED TISSUE SCAFFOLDS

VERFAHREN ZUR HERSTELLUNG VON DEZELLULÄRISIERTEN GEWEBEGERÜSTEN

PROCÉDÉS POUR LA PRODUCTION D'ÉCHAFAUDAGES TISSULAIRES DÉCELLULARISÉS


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 30.07.2015 GB 201513461

(43)Date of publication of application:
06.06.2018 Bulletin 2018/23

(73)Proprietor: UCL BUSINESS LTD
London Greater London W1T 4TP (GB)

(72)Inventors:
  • MAZZA, Giuseppe
    London Greater London NW3 2PF (GB)
  • AL-AKKAD, Walid
    London Greater London NW3 2PF (GB)
  • PINZANI, Massimo
    London Greater London NW3 2PF (GB)

(74)Representative: Mewburn Ellis LLP 
Aurora Building Counterslip
Bristol BS1 6BX
Bristol BS1 6BX (GB)


(56)References cited: : 
US-A1- 2005 249 816
  
  • PETER M CRAPO ET AL: "An overview of tissue and whole organ decellularization processes", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 32, no. 12, 19 January 2011 (2011-01-19), pages 3233-3243, XP028149166, ISSN: 0142-9612, DOI: 10.1016/J.BIOMATERIALS.2011.01.057 [retrieved on 2011-01-25]
  • WANG HUI ET AL: "Decellularization technology in CNS tissue repair", EXPERT REVIEW OF NEUROTHERAPEUTICS, INFORMA UK LTD, UK, vol. 15, no. 5, 1 May 2015 (2015-05-01), pages 493-500, XP009191641, ISSN: 1744-8360, DOI: 10.1586/14737175.2015.1030735
  • REING J E ET AL: "The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 31, no. 33, 1 November 2010 (2010-11-01), pages 8626-8633, XP027381021, ISSN: 0142-9612 [retrieved on 2010-08-21]
  • DENVER M. FAULK ET AL: "Decellularization and Cell Seeding of Whole Liver Biologic Scaffolds Composed of Extracellular Matrix", JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY, vol. 5, no. 1, 1 March 2015 (2015-03-01), pages 69-80, XP055202246, ISSN: 0973-6883, DOI: 10.1016/j.jceh.2014.03.043
  • GIUSEPPE MAZZA ET AL: "Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation", SCIENTIFIC REPORTS, vol. 5, 7 August 2015 (2015-08-07), page 13079, XP055302497, DOI: 10.1038/srep13079
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] This invention relates to the production of extracellular matrix (ECM) scaffolds, for example for use in therapy, disease modelling, drug screening, diagnosis, and discovery of biomarkers.

[0002] Tissue engineering is an emerging field aimed at improving the quality of life for millions of people worldwide by restoring organ function and providing 3D-platforms for studying human disease ex vivo. A key challenge in tissue engineering is the development of 3D-structures ("scaffolds") that recapitulate the physiological architecture and composition of tissues and organs.

[0003] 2-D cell cultures and animal models are commonly used as pre-clinical models for studying human diseases. Although useful information has been obtained from these models, both systems are characterised by several limitations, including: early cellular dedifferentiation and early senescence when in culture, lack of bidirectional tumour-stromal crosstalk, impaired gene and protein expression, lack of correlation with human biology and species-specific toxicity.

[0004] Biological scaffolds composed of extracellular matrix (ECM) are produced by decellularisation of samples of tissue. ECM integrity, bioactivity and 3-D organisation may be preserved in the decellularised scaffolds. However, the production of biological scaffolds is challenging due to the lack of pedicles/vessels that can be cannulated to allow perfusion-decellularisation. Reported protocols are characterised by low speed agitation and require prolonged (e.g. weeks) exposure of the tissue to decellularisation reagents. Furthermore, these protocols are not efficient for complete decellularisation of small-scale tissues and the exterior surface of the tissue is often damaged by the continuous exposure to detergents and enzymes.

[0005] Processes for tissue and whole organ decellularisation are reported in Crapo et al Biomaterials 32 (12) 3233-3243 (2011). The production of a decellularised liver using a series of extractions is reported in US2005/0249816. The use of decellularisation in CNS tissue repair is reported in Wang et al Expert Rev Neurother 15(5) 493-500 (2015). The effects of processing methods on the mechanical and biologic properties of porcine dermal extracellular matrix scaffolds are reported in Reing et al Biomaterials 31 (3) 8626-8633 (2010). The decellularisation and cell seeding of whole liver biologic scaffolds is reported in Faulk et al J Clin Exp Hepatol 2014 5 69-90.

[0006] The present inventors have recognised that a range of different types of tissues may be decellularised without damaging the extracellular matrix using treatment regimens that are characterized by one or more cycles of treatment with sets of different cell damaging agents under high frequency oscillation. These regimes may be useful in the reproducible production of acellular scaffolds that maintain the 3-D architecture and extracellular matrix composition and morphology of the native tissue.

[0007] An aspect of the invention provides a method of producing a decellularised tissue scaffold comprising;
  1. (i) treating a sample of tissue with an osmotic reagent, and
  2. (ii) treating the sample with a detergent,
wherein the tissue sample is subjected to oscillation with a displacement of 1 mm or more and a frequency of 3 to 100 Hz during steps (i) and (ii),
thereby producing a decellularised tissue scaffold.

[0008] A decellularised tissue scaffold produced by the claimed methods consists of acellular extracellular matrix (ECM) from the source tissue and retains the three dimensional architecture, ECM composition and bioactivity of the ECM of the source tissue.

[0009] A decellularised tissue scaffold may be produced using the methods described herein in less than 6 hours, less than 3 hours, or less than 2 hours.

[0010] During treatment with the osmotic reagent, detergent and other decellularisation reagents, the tissue sample is subjected to high frequency oscillation i.e. oscillation of 3 Hz or more. This oscillation enhances the distribution of reagents within the tissue, increases the efficiency of cell lysis and/or improves the flushing of cellular or immunogenic components from the tissue. This reduces the time needed for complete decellularisation, while preserving ECM proteins, 3D-histoarchitecture and the ability of the ECM to induce homing, differentiation and proliferation of cells.

[0011] The oscillation may be in a single plane, preferably in a single linear dimension or line. Any direction or plane of oscillation may be employed. For example, the oscillation may be horizontal (i.e. perpendicular to gravitational force) or vertical (i.e. parallel to gravitational force).

[0012] The sample may be oscillated at a frequency of 3 Hz or more, 5 Hz or more or 10 Hz or more and up to 100 Hz, up to 75 Hz, up to 50 Hz or up to 30 Hz. Suitable ranges of oscillation frequency include any one of the listed lower limits in combination with any of the listed upper limits. For example, the sample may be oscillated at 3 to 75 Hz, for example 10 to 50 Hz.

[0013] The oscillation may subject the sample to a g-force of 4 to 500 ms-2, preferably 40 to 50 ms-2, for example 42ms-2 to 47ms-2. For example, oscillation in any direction may be performed at 1.8 ms-2 to 181.1 ms-2, preferably about 45.3ms-2.

[0014] The displacement of the oscillations may be 1 mm or more, 5 mm or more, 7.5 mm or more, or 10 mm or more, and up to 50 mm, up to 30 mm or up to 25 mmm. Suitable ranges of displacement may include any one of the listed lower limits in combination with any of the listed upper limits. For example, the sample may be oscillated with a displacement of 5 to 50 mm, preferably 7.5 to 25 mm.

[0015] Suitable oscillating motors and other apparatus for oscillating tissue samples are well-known in the art. Suitable means include oscillatory tissue disruptors. For example, tissue samples may be oscillated in a vertical direction using a TissueLyser LT™ device (Qiagen NV, NL) or a horizontal direction using a TissueLyser n™ device (Qiagen, NV, NL).

[0016] Samples of tissue suitable for decellularisation as described herein include sections or blocks of length, width or diameter of 0.1-4 cm, for example 0.2-1.0 cm (e.g. a section with a volume of 0.005 cm3 to 10 cm3, for example 0.008 cm3 to 1 cm3, for example about 0.125 cm3). The section or block may be of any shape. Preferably, the section is of a suitable size for manipulation in standard laboratory vessels, such as multi-well plates, and may be, for example, approximately cubic with sides of about 0.5 cm.

[0017] Small non-vascularized sections or wedges may be useful in reproducing the complexity of 3D tissue microenvironment in small scale for disease modelling and drug screening. Suitable samples may be obtained by punch biopsy, needle biopsy, scalpel cleavage, or using an automatic or non-automatic dicer machine.

[0018] Suitable tissue samples include kidney, muscle, bone, adipose, cartilage, lung, bladder, cornea, skin, liver, intestine, pancreas, prostate, breast, spleen, placenta and heart samples. In some embodiments, the tissue sample may include combination of different tissues, such as an animal tail.

[0019] The tissue sample may be mammalian tissue, for example pig, sheep, rodent, non-human primate or human tissue. Preferably, the tissue sample is human tissue.

[0020] Human tissue for decellularisation may be obtained from human organs that are unsuitable for clinical use in transplantation. Suitable organs may be obtained in accordance with relevant national laws and ethical guidelines. In addition to this, human tissue may be obtained from tissue resection after surgery.

[0021] In some embodiments, the sampled tissue may be normal tissue which does not display pathology associated with damage or disease.

[0022] In other embodiments, the sampled tissue may be pathological tissue which displays pathology associated with damage or disease. For example, sampled tissue may be fatty, fibrotic, cancerous, inflamed or display one or more other features associated with disease or damage. In some embodiments, pathological tissue may display pathology associated with acute or chronic disease, including viral infections, alcohol or toxin damage, fibrosis, amyloidosis and cancer. Examples of pathological tissue include fibrotic, cirrhotic or cancerous liver tissue (from different etiologies), amyloidotic kidney tissue, amyloidotic heart tissue, fibrotic intestine tissue, for example from a patient with Crohn's disease, ulcerative colitis or Inflammatory Bowel Disease (IBD), cancerous pancreatic tissue, fibrotic lung tissue and cancerous breast tissue.

[0023] Decellularised scaffolds produced from pathological tissue samples may have a different structure and composition from scaffolds produced from healthy tissue samples. For example, the morphology of the pathological scaffold or the amounts or relative amounts of ECM components, such as collagen, tenascin and laminin, may be altered in scaffolds from pathological tissue samples compared to healthy tissue samples. Characteristic features of a disease (e.g. amyloidogenic protein) may be associated with the ECM and may be retained during decellularisation, thereby increasing the sensitivity of diagnosis. This may be useful in obtaining specific disease-modified scaffolds for disease modelling, drug screening and diagnosis.

[0024] Pathological tissue may be obtained from an individual with a disease.

[0025] Methods of obtaining and storing tissue and tissue samples for decellularisation as described herein are well-known in the art. For example, the tissue may be heparinized to prevent coagulation and/or perfused with cryoprotectant agents. Suitable cryoprotectants include DMSO, ethylene glycol, propylene glycol, glycerol, 2-methyl-2, 4 pentanediol (MPD), and sucrose.

[0026] The tissue sample is decellularised by a series of sequential exposures to decellularisation reagents, which include osmotic reagents, detergents and optionally proteases and other enzymes, whilst undergoing oscillation. The combination of oscillation and sequential exposure to decellularisation reagents detaches cells and cell debris from the extracellular matrix (ECM) of the tissue sample and removes them without damaging the ECM.

[0027] The tissue sample may be exposed to a decellularisation reagent by immersing the tissue in the reagent. The immersed sample is then oscillated.

[0028] The tissue sample may be oscillated continuously throughout the decellularisation or the oscillation may be stopped to allow removal of the previous decellularisation reagent and addition of the new decellularisation reagent.

[0029] Each step of exposing the tissue sample to a decellularisation reagent may comprise one or more separate treatments with the decellularisation reagent. For example, step (ii) may comprise one, two, three or more separate treatments with an osmotic reagent. Step (iii) may comprise one, two, three or more separate treatments with a detergent.

[0030] The order of exposure to the different decellularisation reagents is based on their different mechanism of actions. Optionally, the cells in the sample may initially be mechanically damaged to promote an intense cellular disruption. The exposure to hypotonic solutions (step ii) amplifies the cell lysis, while washing out cellular materials. The exposure to tissue is exposed to detergent (step iii) to effectively wash out cellular materials.

[0031] A method may comprise repeating step (ii) and/or step (iii) one or more times. For example, the tissue sample may be exposed to multiple cycles of treatment with decellularisation reagents. For example, the tissue sample may be subjected to multiple treatment cycles comprising steps (ii) and (iii) e.g. the tissue sample may be subjected to at least 2, at least 3 or at least 4 treatment cycles comprising steps (ii) and (iii).

[0032] Osmotic stress causes lysis of the cells in the tissue sample and amplifies the effects of the mechanical damage. Osmotic stress may be induced by exposing the tissue sample to one or more osmotic reagents which have a different osmotic pressure to the cells in the tissue (i.e. a non-isotonic reagent). The tissue may be exposed to one or more hypotonic reagents which have a lower osmotic pressure than the cells and subject the cells to a hypotonic environment and/or one or more hypertonic reagents which have a higher osmotic pressure than cells and subjects the cells to a hypertonic environment. Hypotonic reagents may be preferred in some embodiments.

[0033] Hypertonic reagents may be useful, for example, in dissociating DNA from proteins. Suitable hypertonic reagents are well-known in the art and include water, saline (e.g. >0.9% (w/v) NaCl, for example 3% to 10% (w/v) NaCl), which may optionally be buffered for example with phosphate, borate or Tris, polyethylene glycol and dextrose solutions.

[0034] In some preferred embodiments, the osmotic agent is saline, for example 8.7% (w/v) NaCl.

[0035] Hypotonic reagents may be useful for example in inducing cell lysis through simple osmotic effects, with minimal changes in the molecules and architecture of the ECM. Suitable hypotonic reagents are well-known in the art and include deionised water and saline of <0.9% (w/v) NaCl.

[0036] In some preferred embodiments, the osmotic agent is deionised water.

[0037] Detergents solubilise lipids and fats in the tissue and facilitate the removal of cellular debris from the ECM.

[0038] Preferred detergents may include anionic detergents, such as BigCHAP, Bis (polyethylene glycol bis[imidazoyl carbonyl]), Brij®, Brij® 35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL (Sigma, Aldrich), N-Decanoyl- N-methylglucamine, n-Decyl a-D-glucopyranoside, Decyl b-D-maltopyranoside, n-Dodecyl a-D- maltoside, Heptaethylene glycol monodecyl ether, n-Hexadecyl b-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Methyl-6-O-(N-heptylcarbamoyl)-a-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N- Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycolmonooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-b-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycolmonooctyl ether, Polyethylene glycol ether, Polyoxyethylene, Saponin, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85 (Sigma Aldrich), Tergitol, Tetradecyl-b-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monomonotetradecyl ether, Triton® CF-21, Triton® CF-32, Triton® DF-12, Triton® DF-16, Triton® GR-5M, Triton X®- 100, Triton X®-102, Triton X®-15, Triton X®-151, Triton X®-207, Triton®, TWEEN® (Sigma Aldrich), Tyloxapol, n-Undecyl b-D-glucopyranoside, and combinations thereof. Any zwitterionic detergent will work for purposes of the present disclosure. Preferred zwitterionic detergents include, but are not limited to the following: CHAPS, CHAPSO, Sulfobetaine 3-10 (SB 3-10), Sulfobetaine 3-12 (SB 3-12), Sulfobetaine 3-14 (SB 3-14), ASB-14, ASB-16, ASB- C80, Non-Detergent Sulfobetaine (ND SB) 201, DDMAB, DDMAU, EMPIGEN BB®Detergent, 30% Solution, Lauryldimethylamine Oxide (LDAO) 30% solution, ZWITTERGENT® 3-08 Detergent, ZWITTERGENT® 3-10 Detergent, ZWITTERGENT® 3- 12 Detergent, ZWITTERGENT® 3-14 Detergent, ZWITTERGENT® 3-16 Detergent, and combinations thereof.

[0039] Preferred anionic detergents include sodium dodecyl sulfate (SDS) and sodium deoxycholate (SdC). For example, the tissue sample may be exposed to 0.01 to 5% SDS, for example 0.01-1% SDS and/or 0.1 to 10% sodium deoxycholate (SdC), for example about 4% sodium deoxycholate. In some preferred embodiments, the detergent may comprise 4% SDS.

[0040] Detergents may include non-ionic detergents, such as chenodeoxycholic acid, chenodeoxycholic acid sodium salt, cholic acid, ox or sheep bile, dehydrocholic acid, deoxycholic acid, deoxycholic acid methyl ester, digitonin, digitoxigenin, N, N-dimethyldodecylamine N-oxide, docusate sodium salt, glycochenodeoxycholic acid sodium salt, glycocholic acid hydrate, glycocholic acid sodium salt hydrate, glycocholic acid sodium salt, glycolithocholic acid 3-sulfate disodium salt, glycolithocholic acid ethyl ester, N-laurolysarcosine sodium salt, N-laurolysarcosine salt solution, lithium dodecyl sulfate, Lugol solution, niaproof 4, Triton®, Triton® QS-15, Triton® QS-44 solution, 1-octanesulfonic acid sodium salt, sodium 1-butanesulfonate, sodium 1-deccanesulfonate, sodium 1-dodecanesulfonate, sodium 1-heptanesulfonate anhydrous, sodium 1-nonanesulfonate, sodium 1-propanesulfonate monohydrate, sodium 2-bromoethanesulfonate, sodium choleate hydrate, sodium choleate, sodium deoxycholate, sodium deoxycholate monohydrate, sodium dodecyl sulfate, sodium hexanesulfonate anhydrous, sodium octyl sulfate, sodium pentanesulfonate anhydrous, sodium taurocholate, taurochenodeoxycholic acid sodium salt, taurochenodeoxycholic acid sodium salt monohydrate, taurochenodeoxycholic acid sodium salt hydrate, taurolithocholic acid 3-sulfate disodium salt, tauroursodeoxycholic acid sodium salt, Triton X®-200, Triton X®GS-20 solution, trizma dodecyl sulfate, ursodeoxycholic acid, and combinations thereof.

[0041] Preferred non-ionic detergents include polyethylene glycol and Triton™ X-100, e.g. polyethylene glycol p-(1, 1, 3, 3-tetramethylbutyl)-phenyl ether (also called polyoxyethylene octyl phenyl ether or TX-100; CAS 9002-93-1; C14H22O(C2H4O)n. (n = 9-10)). For example, the tissue sample may be exposed to 0.01 to 10% TX-100, or 1 to 3% TX-100, for example 3% TX-100.

[0042] Detergents may include zwitterionic detergents, such as CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), sulfobetaine-10 (3-(Decyldimethylammonio)propanesulfonate), sulfobetaine-16 (n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), and Tri(n-butyl)phosphate. For example, the tissue sample may be exposed to 0.01 to 5% zwitterionic detergent, for example 0.01-1% zwitterionic detergent.

[0043] Numerous other suitable detergents are known in the art and available from commercial sources (e.g. Sigma Aldrich Co LLC, MO, USA) .

[0044] The tissue may be exposed to two or more different detergents. For example, step (iii) may comprise exposing the tissue sample to an anionic detergent, such as SDS and/or SdC and a non-ionic detergent, such as TX-100.

[0045] In some embodiments, the sample may be exposed to SDS, SdC and TX-100, for example 3% SDC; 0.5% SDS and 3% TX-100.

[0046] In some embodiments, the tissue sample may be exposed to a detergent in solution with a chelating agent, such as EDTA or EGTA, which chelate divalent metallic ions, such as Ca2+, and disrupts cell adhesion to the ECM.

[0047] In some embodiments, the tissue sample may be exposed to an alcohol. The alcohol used can be any alcohol, and preferred alcohols are selected from, but are not limited to, the following group: ethyl alcohol, methyl alcohol, n-propyl alcohol, iso-propyl alcohol, n-butyl alcohol, sec-butyl alcohol, t-butyl alcohol, iso-amyl alcohol, n-decyl alcohol and combinations thereof

[0048] In some embodiments, a method of decellularisation may further comprise treating the tissue sample with protease and/or nuclease, for example endonuclease or exonuclease. Preferably, the tissue sample is treated with protease.

[0049] The sample may be treated with protease in a single step or as part of a repeated cycle of treatment with decellularisation agents.

[0050] Proteases degrade proteins in the tissue sample, disrupt cellular and sub-cellular structures and break cell linkages with the ECM. Suitable proteases are well-known in the art and include dispase and trypsin. The tissue sample may be exposed to 0.0025-0.25% (w/v) trypsin, for example 0.025% trypsin.

[0051] In some embodiments, the tissue sample may be exposed to a protease in solution with a chelating agent, such as EDTA, which chelates divalent metallic ions, such as Ca2+, and disrupts cell adhesion to the ECM.

[0052] In preferred embodiments, the tissue sample may be treated with protease and detergent simultaneously.

[0053] For example, the sample may be treated with a solution comprising SDS, SdC, TX-100 and/or Trypsin. These components may be used in any combination or concentration, for example 0.1-10% SdC; 0.1-10% SDS; 0.1-20% TX-100; 0.05-0.5% Trypsin-EDTA, 0.1-10% NaCl, such as 3% SdC; 0.5% SDS; 3% TX-100; 0.025% Trypsin-EDTA, 4.3% NaCl.

[0054] In some embodiments, a method of decellularisation may further comprise treating the tissue sample with peracetic acid (PAA), which denatures DNA and/or ammonium hydroxide, which breaks phosphodiester bonds.

[0055] The tissue sample may be washed following treatment with a decellularisation reagent, for example between one or more of the above steps and/or between cycles.

[0056] The tissue sample may washed with a suitable wash solution, for example a buffered saline, such as phosphate buffered saline (PBS), 0.1-10% saline solution or deionised water. For example, the sample may be washed with 1% PBS after exposure to detergent and optionally protease and/or after exposure to osmotic agent.

[0057] Typically, the tissue sample is washed with 1% PBS for 1 min at ambient temperature.

[0058] In some embodiments, a wash step may comprise two or more sequential exposures to PBS.

[0059] Before steps (ii) and (iii), the cells in the tissue sample may be subjected to mechanical damage to facilitate their removal. The cells may be mechanically damaged by any suitable technique that damages the cells of the tissue without affecting the extracellular matrix, including freeze/thaw, sonication, or high intensity focussed ultrasound (HIFU).

[0060] Preferably, the tissue sample is subjected to at least one cycle of freeze/thaw before decellularisation.

[0061] In some embodiments, mechanical damaging techniques may not be repeated after the initial treatment. For example, such as freeze/thaw treatment after exposure to other decellularisation reagents may lead to ECM damage.

[0062] In other embodiments, the cells in the tissue sample may be subjected to mechanical damaging one or more times after the initial treatment e.g. after one or more repetitions of steps (ii) and/or (iii). For example, the tissue sample may be subjected to HIFU or sonication one or more times.

[0063] Preferably, the cells are mechanically damaged by subjecting the tissue sample to one or more freeze/thaw cycles. For example, the tissue may be frozen at -20°C or less, preferably -50°C or less, - 60°C or less, -70°C or less, and then thawed one or more times. Frozen tissue may be conveniently thawed at 4°C to 37°C. In some embodiments, the tissue may be thawed at about 4°C to minimise temperature gradients within the tissue that may damage the ECM. For example, the tissue may be frozen at about -80°C for 24 hours or more and then thawed at about 4°C. In some preferred embodiments, the tissue sample may be thawed at 37°C, for example for 45 minutes to 1 hour, and then immersed in PBS at 37°C, for example for 15 minutes.

[0064] Tissue samples that are subjected to freeze/thaw are preferably dry to prevent ECM damage. In some embodiments, a tissue sample may be dried before the freeze/thaw step, for example by 5 to 30 minutes exposure at room temperature.

[0065] The tissue sample may be subjected to freeze/thaw in an isotonic buffer, for example saline, such as 0.90% (w/v) NaCl, or PBS.

[0066] The mechanical damage promotes intense cellular disruption within the tissue. The cell lysis caused by the mechanical damage may be amplified by treatment with the osmotic reagent and detergent in steps (ii) and (iii).

[0067] In some embodiments of a first set of embodiments, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes, and
  2. (b) repeating step (a) 0-50 times (e.g. until supernatant is clear)
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes,
  5. (e) repeating step (d),
  6. (f) exposing the sample to saline, e.g. for 2 minutes,
  7. (g) repeating step (f) 0-10 times until tissue is clean of any reagents and protease
  8. (h) repeating steps (a) to (e) 0-10 times (e.g. until tissue is completely white),
  9. (i) exposing the sample to saline, e.g. for 5 minutes,
  10. (j) repeating step (i) 0-10 times.


[0068] In other embodiments of the first set, steps (ii) and (iii) may comprise; may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes, and
  2. (b) repeating step (a) 4 times,
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes,
  5. (e) repeating step (d),
  6. (f) exposing the sample to saline, e.g. for 2 minutes,
  7. (g) repeating step (f) 4 times,
  8. (h) repeating steps (a) to (e).
  9. (i) exposing the sample to saline, for example PBS, e.g. for 5 minutes,
  10. (j) repeating step (i) 2 times.


[0069] Suitable tissue samples include liver, for example human liver, or intestine, for example human intestine.

[0070] A suitable regimen is shown in Table 2 (HL3 and HI5).

[0071] In other embodiments of the first set, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes
  2. (b) repeating step (a) 10 times
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes
  5. (e) repeating step (d)
  6. (f) exposing the sample to saline, e.g. for 2 minutes;
  7. (g) repeating step (f) 4 times
  8. (h) repeating steps (a) to (e).
  9. (i) exposing the sample to saline, for example PBS, e.g. for 5 minutes,
  10. (j) repeating step (i) 2 times


[0072] Suitable tissue samples include liver, for example human liver.

[0073] A suitable regimen is shown in Table 2 (HL43).

[0074] In other embodiments of the first set, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes,
  2. (b) repeating step (d) 11 times,
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes,
  5. (e) repeating step (d),
  6. (f) exposing the sample to saline, e.g. for 2 minutes
  7. (g) exposing the sample to saline, for example PBS, e.g. for 5 minutes,
  8. (h) repeating step (g) 2 times;


[0075] Suitable tissue samples include liver, for example human liver.

[0076] A suitable regimen is shown in Table 2 (HL36).

[0077] In other embodiments of the first set, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes
  2. (b) repeating step (a) 5 to 10 times
  3. (c) exposing the sample to detergent, e.g. for 2 minutes,
  4. (d) repeating step (a) 5 to 10 times,
  5. (e) exposing the sample to saline, for example PBS,
  6. (f) optionally repeating steps (a) to (e) one or more times.


[0078] Suitable tissue samples include kidney, for example human kidney, or heart, for example human heart.

[0079] The sample may be washed between said steps using PBS.

[0080] A suitable regimen is shown in Table 2.

[0081] In the first set of embodiments, the tissue sample may be oscillated vertically at 50 Hz, for example using a TissueLyser LT™.

[0082] In some embodiments of a second set of embodiments, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes,
  2. (b) repeating step (a) 19 times,
  3. (c) exposing the sample to detergent and protease for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes
  5. (e) repeating step (d),
  6. (f) exposing the sample to saline, e.g. for 2 minutes;
  7. (g) repeating step (f) 4 times.
  8. (h) repeating steps (a) to (e) 2 times.
  9. (i) exposing the sample to saline, for example PBS, e.g for 5 minutes,
  10. (j) repeating step (i) 2 times


[0083] A suitable regimen is shown in Table 2 (HL4).

[0084] In other embodiments of the second set, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes, and
  2. (b) repeating step (a) 9 times,
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes,
  5. (e) repeating steps (d),
  6. (f) repeating steps (c) to (e),
  7. (g) exposing the sample to saline, for example PBS, e.g. for 5 minutes, and
  8. (h) repeating step (g) 2 times.


[0085] A suitable regimen is shown in Table 2 (HL43).

[0086] In other embodiments of the second set, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes,
  2. (b) repeating step (a) 9 times,
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes, and
  5. (e) repeating step (d),
  6. (f) exposing the sample to saline for example PBS, e.g. for 5 minutes, and
  7. (g) repeating step (f) 2 times.


[0087] A suitable regimen is shown in Table 2 (HL36).

[0088] In other embodiments of the second set, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes and
  2. (b) repeating step (a) 19 times,
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes
  5. (e) repeating step (d),
  6. (f) exposing the sample to saline, e.g. for 2 minutes
  7. (g) repeating step (f) 5 times,
  8. (h) repeating steps (a) to (d) 5 times.
  9. (i) exposing the sample to saline, for example PBS, e.g. for 5 minutes, and
  10. (j) repeating step (i) 2 times


[0089] A suitable regimen is shown in Table 2 (HL-C1).

[0090] In other embodiments of the second set, steps (ii) and (iii) may comprise;
  1. (a) exposing the sample to deionised water, e.g. for 2 minutes and
  2. (b) repeating step (a) 4 times,
  3. (c) exposing the sample to detergent and protease, e.g. for 2 minutes,
  4. (d) exposing the sample to detergent and protease, e.g. for 4 minutes
  5. (e) repeating step (d)
  6. (f) exposing the sample to peracetic acid (PAA) for 2 minutes
  7. (g) repeating step (f) 1 times
  8. (h) exposing the sample to Ammonium Hydroxide (NH4OH) for 2 minutes
  9. (i) repeating step (h) 2 times
  10. (j) exposing the sample to deionised water, e.g. for 2 minutes and
  11. (k) repeating step (j) 10 times.
  12. (l) repeating steps (c) and (d),
  13. (m) exposing the sample to saline, for example PBS, e.g. for 5 minutes,
  14. (n) repeating step (m) 2 times


[0091] A suitable regimen is shown in Table 2 (HP1).

[0092] In the second set of embodiments, the tissue sample may be oscillated horizontally at 30 Hz, for example using a TissueLyser II™.

[0093] Suitable saline solutions, detergent and protease solutions and other decellularisation reagents are described in more detail above.

[0094] Following decellularisation, the tissue sample may be sterilised, for example by exposure to a sterilising agent. Suitable sterilising agents include γ-irradiation, electrolysed water and chemical agents, such as peracetic acid (PAA). The tissue sample may be exposed to 0.01% peracetic acid and 4% ethanol, for example for 30 minutes to 2 hours. For example, the decellularised tissue sample may be immersed in the sterilising agent and optionally subjected to oscillation, as described herein.

[0095] Following decellularisation and sterilisation, the decellularised tissue sample may be tested, for example for the absence of cells and/or the presence of ECM components, such as collagen, laminin, elastin, proteoglycans, hyaluronic acid, fibronectin, growth factors and extracellular proteases.

[0096] In case of amyloidotic tissue samples, the decellularised tissue sample may be tested, for amyloidotic fibrils.

[0097] Suitable techniques, including macroscopic visualisation, microscopy and immunohistochemical techniques, are well-known in the art.

[0098] Decellularised tissue sample, for example human tissue samples, may lack detectable myofilaments, endothelial cells, smooth muscle cells, and cell debris and nuclei in histologic sections using standard histological staining procedures.

[0099] Decellularised tissue samples produced as described herein preserve the 3-D organ morphology and architecture and the ECM bioactivity of the source tissue sample.

[0100] In some embodiments, the architecture and morphology of a decellularised tissue sample produced by the methods described above may be confirmed by electron microscopy.

[0101] Depending on the source tissue, the decellularised tissue sample may comprise a normal ECM or may be a disease modified ECM. For example, the decellularised tissue sample may comprise one or more structural alterations that are characteristic of a tissue disease or pathology.

[0102] The decellularised tissue samples allow effective attachment, migration, proliferation and three-dimensional organization of cells that are cultured in the scaffold. The decellularised tissue sample may also provide bioactive molecules and bioinductive properties, which maintain cell phenotype and functional properties, and encourage production of tissue specific matrix.

[0103] Following production of decellularised tissue sample as described herein, a method may comprise incubating the sample with a protease such as an elastase or matrix metalloproteinase (MMP). This may be useful in the identification of ECM related biomarkers.
Following production of decellularised tissue sample as described herein, a method may comprise subjecting the decellularised sample to proteomics analysis, for example using electrophoresis and/or mass spectrometry techniques. This may also be useful in the identification of ECM related biomarkers.

[0104] Following production of decellularised tissue scaffold as described herein, a method may comprise re-populating the decellularised scaffold with cells to produce an artificial tissue sample.

[0105] Suitable cells include healthy or diseased cells, such as human primary and cell line tissue cells (e.g. Tissue Sinusoidal Cells), endothelial cells, iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs), mesenchymal stem cells (hMSC), fetal stem cells (e.g. amniotic fluid stem cells), cancer cells, endothelial progenitor cells (EPC) and bipotent liver stem cells.

[0106] Decellularised liver samples may be repopulated with primary hepatocytes, hepatic stellate cells, or Kupffer cells. Decellularised intestine samples may be repopulated with epithelial cells, myofibroblast, endothelial cells, intestinal cancer cells, iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs), mesenchymal stem cells (hMSC) foetal stem cells (e.g. amniotic fluid stem cells) or bipotent liver stem cells. Decellularised pancreatic samples may be repopulated with Islet-beta cells, endothelial cells, pancreatic stellate cells, pancreatic cancer cells, iPSCs or cells derived from patient-specific iPSCs, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) or foetal stem cells (e.g. amniotic fluid stem cells). Decellularised kidney samples may be repopulated with podocytes, tubule cells, MSC, iPSC, foetal stem cells (e.g. amniotic fluid stem cells) or cancer cells. Decellularised heart samples may be repopulated with cardiomyocytes, endothelial cells, iPSC, foetal stem cells (e.g. amniotic fluid stem cells) or MSCs. Decellularised intestinal samples may be repopulated with intestinal stem cells, myofibroblasts, or Caco-2 cells.

[0107] The decellularised tissue sample may be repopulated by seeding the scaffold with cells into the scaffold and culturing under suitable conditions. For example, the cells may be directly injected into the parenchyma of the decellularised scaffold and/or dropped on the surface of the decellularised scaffold. The seeded scaffold may be cultured under static conditions, for example in a culture medium, or under dynamic conditions, for example in a bioreactor.

[0108] In some embodiments, the decellularised tissue scaffold may be repopulated with autologous human cells obtained from a patient, for example to produce artificial tissue for implantation into the patient. In other embodiments, the decellularised human scaffold may be repopulated with allogeneic human cells i.e. cells derived from a different human individual, for example to produce artificial tissue for implantation into the patient. In some embodiments, the allogeneic human cells may be screened for immunocompatibility with the patient before implantation. In other embodiments, the decellularised human scaffold may be repopulated with non-immunogenic cells, for example cell that have been engineered to remove surface antigens, such as HLA, that might elicit an immune response in an individual.

[0109] The disclosure also provides a decellularised tissue scaffold produced by a method described above.

[0110] Decellularised tissue scaffolds produced as described herein are acellular and display the extracellular matrix pore structure, architecture and morphology of the source tissue sample. Decellularised tissue scaffolds produced from fibrotic source tissue samples display the increased ECM components and altered architecture and morphology characteristic of the source tissue.

[0111] The decellularised tissue scaffolds may be useful for disease modelling. Suitable scaffolds may be derived from normal tissue sample or pathological tissue sample, as described above.

[0112] A method of disease modelling may comprise;

providing a decellularised tissue scaffold produced as described above, optionally repopulating the scaffold with cells to produce a re-cellularised tissue, and

determining the effect of a compound, drug, biological agent, device or therapeutic intervention on the scaffold or tissue or the cells therein.



[0113] Methods described herein may be useful in modelling tissue diseases or diseases affecting the tissue, such as tissue fibrosis, tissue cancer and metastases, tissue drug toxicity, post-transplant immune responses, and autoimmune hepatitis.

[0114] The decellularised tissue scaffolds may also be useful for proteomics, biomarker discovery, and diagnostic applications. For example, the effect of a protease on the components, architecture or morphology of a decellularised tissue scaffold may be useful in the identification of biomarkers.

[0115] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0116] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of" and the aspects and embodiments described above with the term "comprising" replaced by the term "consisting essentially of".

[0117] It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0118] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such these are within the scope of the present invention.

[0119] "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0120] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.

Figure 1 shows 5mm3 liver tissue cubes before (top left) and after (bottom left) decellularisation. H&E (Haematoxylin and Eosin) histological staining showed removal of cells after decellularisation. SR (Sirius Red) staining showed preservation of collagen (red) and removal of cellular materials (yellow) in decellularised tissue.

Figure 2 shows immunohistochemistry analysis of extracellular-matrix proteins (ECM). Collagen I, III, IV (structural proteins) were preserved after decellularisation as well as laminin and fibronectin (basement membrane proteins)

Figure 3 shows quantification of DNA. The decellularisation procedure was efficient with a marked decrease in DNA content (p < 0.01).

Figure 4 shows quantitative measurement of collagen after decellularisation. Collagen quantification showed preservation of the amount of collagen in the decellularised tissue when compared to fresh tissue.

Figure 5 shows 150x SEM image including a portal tract surrounded by a typical lobular structure. In addition, SEM image confirmed scaffold acellularity and clearly defined spaces once occupied by hepatocytes (i.e. hepatocyte-free spaces).

Figure 6 shows 5mm3 cirrhotic liver tissue cubes before (top left) and after (bottom left) decellularisation. H&E (Haematoxylin and Eosin) histological staining showed removal of cells after decellularisation. SR (Sirius Red) staining showed preservation of collagen (red) and removal of cellular materials (yellow) in decellularised tissue.

Figure 7 shows quantification of DNA (cirrhotic liver). The decellularisation procedure was efficient with a marked decrease in DNA content (p < 0.01).

Figure 8 shows histological comparison of pancreatic tissue before and after decellularisation. SR (Sirius Red) staining showed preservation of collagen (red) and removal of cellular materials (yellow) in decellularised tissue. H&E (Haematoxylin and Eosin) histological staining showed removal of cells after decellularisation.

Figure 9 shows quantification of DNA (pancreas). The decellularisation procedure was efficient with a marked decrease in DNA content (p < 0.01).

Figure 10 shows histological comparison of human intestine before (left) and after decellularisation (right) showing preservation of collagen structures in all the layers of the tissue after the decellularisation procedure.

Figure 11 shows quantification of DNA (intestine). The decellularisation procedure was efficient with a marked decrease in DNA content (p < 0.01).

Figure 12 shows 3D CAD models of the head (A), centre (B) and base (C) plates produced using Autodesk Inventor Professional 2014 software. Panel (D) depicts the 3D printed, fully assembled cassette. Panels (E) and (F) depict the conjoined base and centre plate and the base plate alone, respectively.


Detailed Description



[0121] An example of a device for decellularising tissue samples as described herein is shown in Figure 12. The device consists of a series of reagent reservoirs (not shown) that house the decellularisation reagents required during process operation. The reservoirs connect to a cassette 2 via tubing (not shown). The tubing is associated with a reagent driving pump (not shown). The reagents enter the cassette 2 via an inlet port 7 that is situated in a base plate 3 of the cassette 2, before travelling along an inflow conduit 8 and being equally divided between multiple sample chambers 6 located in a centre plate 4 of the cassette 2. To enter the sample chambers 6, decellularisation reagents must either flow through appropriate membranes (not shown) and/or past extrusions 12 that retain the sample within the chambers 6 located in the centre plate 4. The entire cassette 2 is then oscillated by the oscillating motor (not shown), in accordance with the decellularisation protocol. Reagents are then drained from the cassette 2 via the outflow conduit 9 in the base plate, before exiting the cassette through the outlet port 10 and progressing toward the external waste reservoir (not shown) via tubing and associated driving pump(s) (not shown). During draining, the removed reagents are replaced with filtered air that enters the cassette via a series of conduits in the head plate 5 of the cassette 2. Reagents are prevented from entering these conduits via hydrophobic membranes (not shown) and/or extrusions 20 in the head plate 5. During stationary phases of the process, the reagents and/or tissues may be sampled via integrated sampling ports (not shown) for off-line process assays. The whole process is controlled via a processor (not shown) that allows for control of all aspects of the process, both predetermined and in response to feedback loops.

Experiments


1. Determining G force



[0122] The novel system used for decellularising biological tissue is considered to be a simple harmonic oscillator as there is only one force acting on the system in a periodic motion where the restoring force is directly proportional to the displacement and acts in the direction opposite to that of the displacement, with a constant amplitude and frequency. From this statement we can build an equation, which will allow us to calculate the G-force experienced by the tissue and reagents during decellularisation. Therefore, a biological tissue undergoing harmonic motion with frequency f Hz and amplitude Û m and assuming it starts at zero displacement (i.e. x=0 and t=0) will occupy the position x = Ûsin (f*2*π*t) (considering the difference in density between the tissue and it surrounding solution is negligible). Furthermore, a particle (or tissue in this case) which moves under simple harmonic motion will have the equation x"= -w2x, where w=2*pi*f. Substituting these equation into Newton second law, Force (F) equals mass (m) multiplied by acceleration (a) [F=ma], results in the equation F = -m[(2*π*f)2]*Ûsin(f*2*π*t). This is extremised when |sin|=1. Therefore, it will be at its maximum/minimum when F = +/-Ûm[(2*π*f)2]. Finally by dividing the equation by the force experienced by gravity (mg), the equation for G-force = (Û/g) * [(2*π*f) 2].

2. Reagents



[0123] 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.

[0124] Sodium deoxycholate solution (SDS) 4%: 40g of Sodium deoxycholate, BioXtra, ≥98.0% (Sigma-Aldrich) is added to 1L of deionized water (MilliQ by Millipore), and stirred using a magnetic stirrer for 1 hr.

[0125] Saline Solution 8.7%: 87 g of Sodium Chloride, >99% (Sigma-Aldrich) is added to 1L of deionized water (MilliQ by Millipore), and stirred using a magnetic stirrer for 1 hr.

[0126] Reagent Mixture Solution (SDC3%; SDS 0,5%; TX1000.3%; T/E0.025%; NaCl 4.3%): 30g of Sodium deoxycholate, BioXtra, ≥98.0% (Sigma-Aldrich), 5 g of Sodium dodecyl sulfate, BioXtra, ≥99.0 (Sigma-Aldrich), 3 ml of Triton X-100 (Sigma-Aldrich), 10ml of 0.25% Gibco® Trypsin-EDTA (Life technologies) and 43g of Sodium Chloride (Sigma-Aldrich) are added to deionized water (MilliQ by Millipore) to make a total of 1 L and stirred using a magnetic stirrer.

[0127] Peracetic acid (PAA) 0.1 %: 1 ml of Peractic acid solution, purum, ∼39% in acetic acid (Sigma-Aldrich) is added to 1L of deionized water (MilliQ by Millipore), and stirred using a magnetic stirrer for 1 hr.

[0128] Ammonium Hydroxide (NH4OH) 0.1 %: 1ml of Ammonium hydroxide solution, ACS reagent, 28.0-30.0% NH3 basis (Sigma-Aldrich) is added to 1L of deionized water (MilliQ by Millipore), and stirred using a magnetic stirrer for 1 hr.

3. Methods


3.1 Tissue decellularisation



[0129] The protocols for decellularising biological tissue are described elsewhere herein and shown in Tables 2 and 3.

[0130] After the completion of a protocol, random samples are selected and; fixed in 10% formalin for histological and immunohistochemistry studies, snap-frozen in liquid nitrogen and stored at -80 °C freezer until needed for the DNA, collagen and elastin quantification assays, fixed in 2.5 Glutaraldehyde for SEM imaging or stored in 1% PBS at 4°C until needed for bioengineering experiments.

[0131] Initially liver cubes are thawed in a water bath at 37 °C for 1 hour (hr), followed by the addition of 1.2 ml of 1% PBS for 15 minutes (mins). Once thawed the cubes are transferred into 2ml safe-lock tubes (Eppendorf). A standardised 1.5 ml of each solution is added to its respected tube/protocol.

3.2 Histology and Immunohistochemistry



[0132] Tissue samples, previously fixed in 10% formalin, were retrieved, washed in distilled water, dehydrated in a series of Industrial Denatured Alcohol (IDA) (Acquascience) and xylene baths and finally embedded in paraffin. The samples were then sliced into 5 µm sections using a Leica RM2035 microtome (Leica Biosystems). All sections were then passed through three histology grade xylene (Acquascience) baths for a minimum of 5 mins, and then through three IDA (Acquascience) baths for a minimum of 2 mins, finally ending up in tap water.

3.3 Histology



[0133] Sections were stained at room temperature as follows:
Haematoxylin and Eosin: Sections were treated with haematoxylin Harris' formula (Leica biosystems) for 10 mins and then washed in tap water for 5 mins. Next, the sections were stained with eosin (Leica biosystems) for 3 mins, and then washed again with water. The sections were then dehydrated through IDA (Industrial Denatured Alcohol) (Acquascience) as quickly as possible and then placed in histology grade xylene (Acquascience) until mounted.

[0134] Pico-Sirius Red: Sections were treated with freshly filtered pico-sirius red - F38 (R.A.Lamb; CI-35780) for 20 mins. The section were then dehydrated through IDA (Acquascience) as quickly as possible and then placed in histology grade xylene (Acquascience) until mounted.

[0135] Elastic Van Gieson: Sections were treated with 0.5% potassium permanganate for 5 mins and washed thoroughly with distilled water. Next, they were treated with 1% oxalic acid for 1 minute, washed with distilled water followed by absolute alcohol. Sections were then stained with neat Miller's Elastic - (R.A. Lamb; LAMB/080D) for 2 hrs, washed thoroughly with 70% industrially methylated spirits (IMS) (Fisher scientific) and then placed in tap water. The sections were checked under the microscope and, if necessary, differentiated in 0.5% acid-alcohol (1% HCl in 70% IDA aq.). As a final step, the sections were stained with van Gieson (Leica biosystems) for 5 mins. The section were then dehydrated through IDA (Acquascience) as quickly as possible and then placed in histology grade xylene (Acquascience) until mounted.

3.4 Immunohistochemistry



[0136] Slides were incubated in 0.5% Trypsin (MP Biomedical) / 0.5% Chymotrypsin (Sigma) / 1% Calcium Chloride (BDH) in 10% Tris buffered saline (TBS) for 30 minutes at 37 °C. Slides were then washed in 10% TBS at pH 7.6 with 0.04% Tween-20 (Sigma) for 5 mins. The slides were later blocked in peroxide blocking solution (Novocastra) for 5 minutes and 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) and laminin (mouse mAb to laminin α5-chain (MAB1924), diluted 1:200; Millipore). The slides were then placed for 25 minutes in Novolink™ post primary (Novocastra), 25 mins in Novolink™ polymer solution (Novocastra) and developed with Novolink™ 3, 3' di-amino-benzidine (Novocastra). The slides were finally counterstained with Mayer's Haematoxylin (Sigma) for 1 mins.

[0137] All sections 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).

3.5 DNA quantification



[0138] Decellularised tissue cubes used for all protocols were retrieved from the -80 °C freezer and thawed in a 37 °C water bath for 1 hr. The liver cubes were then weighed and if necessary, cut to be between 15 and 25 mg in weight. The cubes were then placed in 1.5 ml microcentrifuge tubes. Twenty µl of proteinase K was added to each, and then mixed thoroughly using a vortex. The cubes were then placed into a heating block at 56 °C for at least 16 hrs or until cubes were completely lysed. The DNA was then extracted using the QIAGEN DNAeasy Blood and Tissue Kit according to the manufacturer's instructions. The extracted DNA was eluted in 200 µl of buffer AE and was quantified using a NanoDrop ND-2000 spectrophotometer.

3.6 Collagen quantification



[0139] 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.

3.7 Scanning Electron Microscopy (SEM)



[0140] 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 CO2 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 2nm thick) using a Gatan ion beam coater. Images were recorded with a 7401 FEG scanning electron microscope (Jeol, USA)

3.8 Bioengineering procedure



[0141] Biological scaffolds were kept overnight in complete medium [day - 1]. Cells were re-suspended at a concentration of 2 million cells per 50 µl (2x106/50µL) 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 decellularised tissue. Seeded scaffolds were kept for 2h in a humidified environment at 37 °C with 5% CO2 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.

4. Results



[0142] Decellularised healthy liver cubes (HL3, HL4, HL36 and HL43) were all macroscopically translucent and white in colour (Figure 1). The vascular network was also visible and the tissue maintained its cubic structure. In addition, both native and decellularised liver tissue were inspected histologically for nuclear material (H&E staining) and cellular remnants (SR staining). The decellularisation protocols were successful at eliminating all cellular material (Figure 1). This was confirmed by quantification the amount of DNA remaining in the decellularised liver cubes, which was significantly lower (p<0.001) that that of fresh liver tissue (Figure 3).

[0143] To investigate the retention of ECM proteins, immunohistochemistry was performed. Five ECM proteins were investigated, collagen I, collagen III, collagen IV, fibronectin and laminin (Figure 2). Collagen I and III can be seen lining the portal tract area, while collagen IV and fibronectin was evident within the liver lobules. Laminin staining was positive around the vessels and bile duct. Similarly, collagen quantification demonstrated that the decellularised liver cubes were able to preserve collagen when compared to fresh tissue (Figure 4).

[0144] Furthermore, the decellularised liver cubes were analysed by scanning electron microscopy (SEM). 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 fibers structuring the hepatocyte-free spaces, as well as portal tracts and lobular structure, were found to be an exceptionally preserved (Figure 5). To further investigate the mechanical properties of the scaffolds, the stiffness of the native tissue and decellularised tissue were measured using atomic force microscopy (AFM). This revealed no significant difference in stiffness between native and decellularised tissue.

[0145] Decellularised liver scaffolds were repopulated with different types of human liver parenchymal and non-parenchymal cells. These cells were found to exhibit excellent viability, motility and remodelling of the extracellular matrix. Furthermore, bioengineered scaffolds showed remarkable difference in gene expression when compared with standard 2D-system.

[0146] Decellularised cirrhotic liver tissue (HLC1) was similarly white in colour. Likewise, the vascular network was also visible and the tissue maintained its cubic structure. In addition, both native and decellularised cirrhotic liver tissue was inspected histologically for nuclear material (H&E staining) and cellular remnants (SR staining). The decellularisation protocols were successful at eliminating all cellular material (Figure 6). This was confirmed by quantification the amount of DNA remaining in the decellularised liver cubes, which was significantly lower (p<0.001) that that of fresh cirrhotic liver tissue (Figure 7).

[0147] The decellularised cirrhotic liver tissue was also able to retain the distorted hepatic architecture that characterizes fibrotic tissue. This can be seen in both the SR and H&E staining, which show well preserved nodules and fibrotic septa.

[0148] Decellularised pancreatic tissue was inspected histologically for nuclear material (H&E staining) and cellular remnants (SR staining). The decellularisation protocols were successful at eliminating most cellular material (Figure 8). This was confirmed by quantification the amount of DNA remaining in the decellularised pancreatic cubes, which was significantly lower (p<0.001) that that of fresh pancreatic tissue (Figure 9). The pancreatic architecture was also preserved. Both SR and H&E staining clearly show the preservation of the ECM where the Islets of Langerhans were situated (Figure 8).

[0149] Finally, decellularised intestinal tissue was inspected histologically for cellular remnants (SR staining). The decellularisation protocols were successful at eliminating all cellular material (Figure 10). This was confirmed by quantification the amount of DNA remaining in the decellularised intestinal cubes, which was significantly lower (p<0.001) that that of fresh intestinal tissue (Figure 11). The intestinal microarchitecture was also preserved. SR staining visibly show the preservation of all four intestinal layers; villous mucosa, submucosa, muscularis externa and adventitia (Figure 10).

Table 2
Tissue IDProtocolTime (mins)
HL3 (125mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 5 times) 66
2. RM, 2 mins
3. RM 4 mins (repeat a total of 2 times)
4. PBS 1%, 1 min
5. Saline Solution 8.7%, 2 mins (repeat a total of 5 times)
6. Repeat steps 1-3
7. PBS 1%, 5 mins (repeat a total of 3 times)
HL43 (125mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 11 times) 76
2. RM, 2 mins
3. RM 4 mins (repeat a total of 2 times)
4. PBS 1%, 1 min
5. Saline Solution 8.7%, 2 mins (repeat a total of 5 times)
6. Repeat steps 1-3
7. PBS 1%, 5 mins (repeat a total of 3 times)
HL36 (125mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 12 times) 42
2. RM, 2 mins
3. RM 4 mins (repeat a total of 2 times)
4. PBS 1%, 1 min
5. Saline Solution 8.7%, 2 mins
6. PBS 1%, 5 mins (repeat a total of 3 times)
HI5 (60-75mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 5 times) 66
2. RM, 2 mins
3. RM 4 mins (repeat a total of 2 times)
4. PBS 1%, 1 min
5. Saline Solution 8.7%, 2 mins (repeat a total of 5 times)
6. Repeat steps 1-3
7. PBS 1%, 5 mins (repeat a total of 3 times)
Kidney (bio psy; 15.5mm3 ) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 5-10 times)  
2. SDC 4% 2 mins (repeat a total of 5-10 times)
3. PBS 2 mins
These steps may be repeated one or more times
Heart (biop sy; 8mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 5-10 times)  
2. SDC 4% 2 mins (repeat a total of 5-10 times)
3. PBS 2 mins
These steps may be repeated one or more times
Table 3
Tissue IDProtocolTime (mins)
HL4 (216mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 20 times) 116
  2. RM, 2 mins  
  3. RM 4 mins (repeat a total of 2 times)  
  4. PBS 1%, 1 min  
  5. Saline Solution 8.7%, 2 mins (repeat a total of 5 times)  
  6. Repeat steps 1-3 (repeat a total of 2 times)  
  7. PBS 1%, 5 mins (repeat a total of 3 times)  
HL43 (125mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 10 times) 56
  2. RM, 2 mins  
  3. RM 4 mins (repeat a total of 2 times)  
  4. PBS 1%, 1 min  
  5. Repeat steps 2-3  
  6. PBS 1%, 5 mins (repeat a total of 3 times)  
HL36 (125mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 10 times) 45
  2. RM, 2 mins  
  3. RM 4 mins (repeat a total of 2 times)  
  4. PBS 1%, 1 min (repeat a total of 3 times)  
HL-C1 (125mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 20 times) 166
2. RM, 2 mins  
  3. RM 4 mins (repeat a total of 2 times)  
  4. PBS 1%, 1 min  
  5. Saline Solution 8.7%, 2 mins (repeat a total of 5 times)  
  6. Repeat steps 1-3 (repeat a total of 2 times)  
  7. PBS 1%, 5 mins (repeat a total of 3 times)  
  8. Repeat step 2-3  
HP1 (125mm3) 1. Deionised Water (MilliQ), 2 mins (repeat a total of 5 times) 79
2. RM, 2 mins  
  3. RM 4 mins (repeat a total of 2 times)  
  4. PBS 1%, 1 min(repeat a total of 2 times)  
  5. PAA 0.1%, 2 min (repeat a total of 2 times)  
  6. NH4OH 0.1%, 2 min (repeat a total of 3 times)  
  7. Deionised Water (MilliQ), 2 mins (repeat a total of 10 times)  
  8. Repeat steps 2-3  
  9. PBS 1%, 5 mins (repeat a total of 3 times)  



Claims

1. A method of producing a decellularised tissue scaffold comprising;

(i) treating a sample of tissue with an osmotic reagent, and

(ii) treating the sample with a detergent,

wherein the tissue sample is subjected to oscillation with a displacement of 1 mm or more and a frequency of 3 to 100 Hz during steps (i) and (ii),
thereby producing a decellularised tissue scaffold.
 
2. The method according to claim 1 wherein the sample is subjected to oscillation at 3 to 75 Hz.
 
3. The method according to any one of the preceding claims wherein the oscillation subjects the tissue sample to a g-force of 4 to 500 ms-2.
 
4. The method according to any one of the preceding claims wherein the oscillation has a displacement of 5 to 50 mm.
 
5. The method according to any one of the preceding claims wherein the tissue sample is kidney, muscle, bone, adipose, cartilage, lung, bladder, cornea, skin, liver, spleen, placenta, intestine, pancreas, prostate, breast or heart.
 
6. The method according to any one of the preceding claims wherein the osmotic agent is a hypotonic agent, optionally deionised water.
 
7. The method according to any one of the preceding claims wherein the detergent comprises polyethylene glycol p-(1, 1, 3, 3-tetramethylbutyl)-phenyl ether (Triton X100™), SDS, and/or SdC.
 
8. The method according to any one of the preceding claims wherein the tissue sample is treated with the detergent in combination with a protease, optionally trypsin.
 
9. The method according to any one of the preceding claims wherein the cells in the tissue sample are mechanically damaged in step (i), optionally by freezing and thawing the tissue sample.
 
10. The method according to any one of the preceding claims comprising any one of;

A)

(a) exposing the sample to deionised water,

(b) repeating step (a) 0 to 50 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes,

(e) repeating step (d),

(f) exposing the sample to saline,

(g) repeating step (f) 0-10 times,

(h) repeating steps (a) to (e) 0-10 times,

(i) exposing the sample to saline, and

(j) repeating step (i) 0-10 times; or

B)

(a) exposing the tissue sample to deionised water,

(b) repeating step (a) 4 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes,

(e) repeating step (d),

(f) exposing the sample to saline,

(g) repeating step (e) 4 times,

(h) repeating steps (a) to (e),

(i) exposing the sample to saline, and

(j) repeating step (i) 2 times; or

c)

(a) exposing the tissue sample to deionised water,

(b) repeating step (a) 10 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes,

(e) repeating step (d),

(f) exposing the sample to saline,

(g) repeating step (f) 4 times,

(h) repeating steps (a) to (e),

(i) exposing the sample to saline, and

(j) repeating step (i) 2 times; or

D)

(a) exposing the sample to deionised water,

(b) repeating step (a) 11 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes,

(e) repeating step (d),

(e) exposing the sample to saline for 2 minutes,

(f) exposing the sample to saline for 5 minutes, and

(g) repeating step (f) 2 times;

optionally wherein the tissue sample is a liver sample.


 
11. The method according to any one of claims 1 to 10 comprising;

(a) exposing the sample to deionised water,

(b) repeating step (a) 5 to 10 times,

(c) exposing the sample to detergent,

(d) repeating step (a) 5 to 10 times,

(e) exposing the sample to saline,

(f) optionally repeating steps (a) to (e) one or more times; optionally wherein the tissue sample is a kidney or heart sample.


 
12. The method according to claim 11 wherein the tissue sample is oscillated vertically at 50 Hz.
 
13. The method according to any one of claims 1 to 10 comprising any one of;

A)

(a) exposing the sample to deionised water,

(b) repeating step (a) 19 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 2 minutes,

(e) repeating step (d),

(f) exposing the sample to saline

(g) repeating step (f) 4 times,

(h) repeating steps (a) to (e) 2 times,

(i) exposing the sample to saline,

(j) repeating step (i) 2 times; or

B)

(a) exposing the sample to deionised water,

(b) repeating step (a) 9 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes

(e) repeating step (d)

(f) repeating steps (c) to (e),

(g) exposing the sample to saline,

(h) repeating step (g) 2 times; or

C)

(a) exposing the sample to deionised water,

(b) repeating step (a) 10 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes,

(e) repeating step (d),

(f) exposing the sample to saline, and

(g) repeating step (f) 2 times; or

D)

(a) exposing the sample to deionised water

(b) repeating step (a) 20 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes

(e) repeating step (d),

(f) exposing the sample to saline,

(g) repeating step (f) 5 times,

(h) repeating steps (a) to (d) 5 times,

(i) exposing the sample to saline, and

(j) repeating step (i) 2 times; or

E)

(a) exposing the sample to deionised water,

(b) repeating step (a) 4 times,

(c) exposing the sample to detergent and protease for 2 minutes,

(d) exposing the sample to detergent and protease for 4 minutes,

(e) repeating step (d),

(f) exposing the sample to peracetic acid (PAA),

(g) repeating step (f) 1 times,

(h) exposing the sample to ammonium hydroxide (NH4OH),

(i) repeating step (h) 2 times,

(j) exposing the sample to deionised water,

(k) repeating step (j) 10 times,

(l) repeating steps (c) and (d),

(m) exposing the sample to saline,

(n) repeating step (m) 2 times;

optionally wherein the tissue sample is a liver, pancreas or intestine sample.


 
14. The method according to claim 13 wherein the tissue sample is oscillated horizontally at 30 Hz.
 
15. The method according to any one of the preceding claims wherein the tissue sample is exposed to the osmotic agent and/or detergent for 2 to 4 minutes in each repetition of steps (ii) and (iii).
 
16. The method according to any one of claims 1 to 15 comprising

(i) sterilising the scaffold following decellularisation and/or

(ii) repopulating the decellularised scaffold with cells to produce a recellularised scaffold.


 
17. A method of disease modelling comprising;
producing a decellularised tissue scaffold by a method according to any one of claims 1 to 15, optionally repopulating the sample with cells to produce a recellularised tissue, and,
determining the effect of a compound, drug, biological agent, device or therapeutic intervention on the scaffold.
 
18. A method of identifying an ECM biomarker comprising;
producing a decellularised tissue scaffold by a method according to any one of claims 1 to 15,
exposing the scaffold to a protease, and
determining the degradation of one or more components of the scaffold by the protease, and
identifying one or more components degraded by the protease as ECM biomarkers.
 
19. A method of diagnosing the presence of a diseased tissue in an individual comprising;
producing a decellularised scaffold by a method according to any one of claims 1 to 15 from a tissue of the individual, and
determining the structure and/or composition of the scaffold,
wherein aberrant structure and/or composition of the scaffold is indicative that the tissue is diseased in the individual.
 


Ansprüche

1. Verfahren zur Herstellung eines dezellularisierten Gewebegerüsts, das Folgendes umfasst:

(i) Behandeln einer Gewebeprobe mit einem osmotischen Reagens und

(ii) Behandeln der Probe mit einem Tensid,

wobei die Gewebeprobe während der Schritte (i) und (ii) Oszillation mit einer Verschiebung von 1 mm oder mehr und einer Frequenz von 3 bis 100 Hz unterzogen wird, wodurch ein dezellularisiertes Gewebegerüst hergestellt wird.
 
2. Verfahren nach Anspruch 1, wobei die Probe Oszillation mit 3 bis 75 Hz unterzogen wird.
 
3. Verfahren nach einem der vorangegangenen Ansprüche, wobei die Gewebeprobe durch die Oszillation einer g-Kraft von 4 bis 500 m.s-2 ausgesetzt wird.
 
4. Verfahren nach einem der vorangegangenen Ansprüche, wobei die Oszillation eine Verschiebung von 5 bis 50 mm aufweist.
 
5. Verfahren nach einem der vorangegangenen Ansprüche, wobei die Gewebeprobe eine Nieren-, Muskel-, Knochen-, Fett-, Knorpel-, Lungen-, Blasen-, Hornhaut-, Haut-, Leber-, Milz-, Plazenta-, Darm-, Pankreas-, Prostata-, Brust- oder Herzprobe ist.
 
6. Verfahren nach einem der vorangegangenen Ansprüche, wobei das osmotische Mittel ein hypotonisches Mittel, gegebenenfalls entionisiertes Wasser, ist.
 
7. Verfahren nach einem der vorangegangenen Ansprüche, wobei das Tensid Polyethylenglykol-p-(1,1,3,3-tetramethylbutyl)phenylether (Triton X100™), SDS und/oder SdC umfasst.
 
8. Verfahren nach einem der vorangegangenen Ansprüche, wobei die Gewebeprobe mit dem Tensid in Kombination mit einer Protease, gegebenenfalls Trypsin, behandelt wird.
 
9. Verfahren nach einem der vorangegangenen Ansprüche, wobei die Zellen in der Gewebeprobe in Schritt (i) mechanisch, gegebenenfalls durch Einfrieren und Auftauen der Gewebeprobe, geschädigt werden.
 
10. Verfahren nach einem der vorangegangenen Ansprüche, das Folgendes umfasst:

A)

(a) Aussetzen der Probe gegenüber entionisiertem Wasser,

(b) 0- bis 50-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Aussetzen der Probe gegenüber Kochsalzlösung,

(g) 0- bis 10-maliges Wiederholen von Schritt (f),

(h) 0- bis 10-maliges Wiederholen der Schritte (a) bis (e),

(i) Aussetzen der Probe gegenüber Kochsalzlösung und

(j) 0- bis 10-maliges Wiederholen von Schritt (i); oder

B)

(a) Aussetzen der Gewebeprobe gegenüber entionisiertem Wasser,

(b) 4-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Aussetzen der Probe gegenüber Kochsalzlösung,

(g) 4-maliges Wiederholen von Schritt (e),

(h) Wiederholen der Schritte (a) bis (e),

(i) Aussetzen der Probe gegenüber Kochsalzlösung und

(j) 2-maliges Wiederholen von Schritt (i); oder

C)

(a) Aussetzen der Gewebeprobe gegenüber entionisiertem Wasser,

(b) 10-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Aussetzen der Probe gegenüber Kochsalzlösung,

(g) 4-maliges Wiederholen von Schritt (f),

(h) Wiederholen der Schritte (a) bis (e),

(i) Aussetzen der Probe gegenüber Kochsalzlösung und

(j) 2-maliges Wiederholen von Schritt (i); oder

D)

(a) Aussetzen der Probe gegenüber entionisiertem Wasser,

(b) 11-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(e) 2-minütiges Aussetzen der Probe gegenüber Kochsalzlösung,

(f) 5-minütiges Aussetzen der Probe gegenüber Kochsalzlösung und

(g) 2-maliges Wiederholen von Schritt (f);

wobei die Gewebeprobe gegebenenfalls eine Leberprobe ist.


 
11. Verfahren nach einem der Ansprüche 1 bis 10, das Folgendes umfasst:

(a) Aussetzen der Probe gegenüber entionisiertem Wasser,

(b) 5- bis 10-maliges Wiederholen von Schritt (a),

(c) Aussetzen der Probe gegenüber Tensid,

(d) 5- bis 10-maliges Wiederholen von Schritt (a),

(e) Aussetzen der Probe gegenüber Kochsalzlösung,

(f) gegebenenfalls ein- oder mehrmaliges Wiederholen der Schritte (a) bis (e); wobei die Gewebeprobe gegebenenfalls eine Nieren- oder Herzprobe ist.


 
12. Verfahren nach Anspruch 11, wobei die Gewebeprobe mit 50 Hz vertikal oszilliert wird.
 
13. Verfahren nach einem der Ansprüche 1 bis 10, das Folgendes umfasst:

A)

(a) Aussetzen der Probe gegenüber entionisiertem Wasser,

(b) 19-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Aussetzen der Probe gegenüber Kochsalzlösung,

(g) 4-maliges Wiederholen von Schritt (f),

(h) 2-maliges Wiederholen der Schritte (a) bis (e),

(i) Aussetzen der Probe gegenüber Kochsalzlösung und

(j) 2-maliges Wiederholen von Schritt (i); oder

B)

(a) Aussetzen der Gewebeprobe gegenüber entionisiertem Wasser,

(b) 9-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Wiederholen der Schritte (c) bis (e),

(g) Aussetzen der Probe gegenüber Kochsalzlösung,

(h) 2-maliges Wiederholen von Schritt (g); oder

C)

(a) Aussetzen der Probe gegenüber entionisiertem Wasser,

(b) 10-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Aussetzen der Probe gegenüber Kochsalzlösung und

(g) 2-maliges Wiederholen von Schritt (f); oder

D)

(a) Aussetzen der Probe gegenüber entionisiertem Wasser,

(b) 20-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Aussetzen der Probe gegenüber Kochsalzlösung,

(g) 5-maliges Wiederholen von Schritt (f);

(h) 5-maliges Wiederholen der Schritte (a) bis (d),

(i) Aussetzen der Probe gegenüber Kochsalzlösung und

(j) 2-maliges Wiederholen von Schritt (i); oder

(E)

(a) Aussetzen der Probe gegenüber entionisiertem Wasser,

(b) 4-maliges Wiederholen von Schritt (a),

(c) 2-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(d) 4-minütiges Aussetzen der Probe gegenüber Tensid und Protease,

(e) Wiederholen von Schritt (d),

(f) Aussetzen der Probe gegenüber Peressigsäure (PAA),

(g) 1-maliges Wiederholen von Schritt (f),

(h) Aussetzen der Probe gegenüber Ammoniumhydroxid (NH4OH),

(i) 2-maliges Wiederholen von Schritt (h),

(j) Aussetzen der Probe gegenüber entionisiertem Wasser,

(k) 10-maliges Wiederholen von Schritt (j),

(l) Wiederholen der Schritte (c) und (d),

(m) Aussetzen der Probe gegenüber Kochsalzlösung,

(n) 2-maliges Wiederholen von Schritt (m);

wobei die Gewebeprobe gegebenenfalls eine Leber-, Pankreas- oder Darmprobe ist.


 
14. Verfahren nach Anspruch 13, wobei die Gewebeprobe mit 30 Hz horizontal oszilliert wird.
 
15. Verfahren nach einem der vorangegangenen Ansprüche, wobei die Gewebeprobe dem osmotischen Mittel und/oder Tensid bei jeder Wiederholung der Schritte (i) und (ii) 2 bis 4 Minuten lang ausgesetzt wird.
 
16. Verfahren nach einem der Ansprüche 1 bis 15, das Folgendes umfasst:

(i) Sterilisieren des Gerüsts nach Dezellularisierung und/oder

(ii) Neubesiedeln des dezellularisierten Gerüsts mit Zellen, um ein rezellularisiertes Gerüst herzustellen.


 
17. Verfahren zur Krankheitsmodellierung, das Folgendes umfasst:

Herstellen eines dezellularisierten Gewebegerüsts durch ein Verfahren nach einem der Ansprüche 1 bis 15, gegebenenfalls Neubesiedeln der Probe mit Zellen, um ein rezellularisiertes Gewebe herzustellen, und

Bestimmen der Wirkung einer Verbindung, eines Arzneimittels, eines biologischen Wirkstoffs, einer Vorrichtung oder eines therapeutischen Eingriffs auf das Gewebe.


 
18. Verfahren zur Identifikation eines ECM-Biomarkers, das Folgendes umfasst:

Herstellen eines dezellularisierten Gewebegerüsts durch ein Verfahren nach einem der Ansprüche 1 bis 15,

Aussetzen des Gerüsts gegenüber einer Protease und

Bestimmen des Abbaus einer oder mehrerer Komponenten des Gerüsts durch die Protease, sowie

Identifizieren einer oder mehrerer Komponenten, die durch die Protease abgebaut wurden, als ECM-Biomarker.


 
19. Verfahren zum Diagnostizieren des Vorliegens eines erkrankten Gewebes in einem Individuum, das Folgendes umfasst:

Herstellen eines dezellularisierten Gerüsts durch ein Verfahren nach einem der Ansprüche 1 bis 15 aus einem Gewebe des Individuums und

Bestimmen der Struktur und/oder Zusammensetzung des Gerüsts,

wobei eine abweichende Struktur und/oder Zusammensetzung des Gerüsts ein Indikator dafür ist, dass das Gewebe in dem Individuum erkrankt ist.
 


Revendications

1. Procédé de production d'un échafaudage tissulaire décellularisé comprenant ;

(i) le traitement d'un échantillon de tissu avec un réactif osmotique, et

(ii) le traitement de l'échantillon avec un détergent, dans lequel l'échantillon de tissu est soumis à oscillation avec un déplacement de 1 mm ou plus et une fréquence de 3 à 100 Hz pendant les étapes (i) et (ii),

de façon à produire un échafaudage tissulaire décellularisé.
 
2. Procédé selon la revendication 1 dans lequel l'échantillon est soumis à une oscillation à 3 à 75 Hz.
 
3. Procédé selon l'une quelconque des revendications précédentes dans lequel l'oscillation soumet l'échantillon de tissu à une force g de 4 à 500 ms-2.
 
4. Procédé selon l'une quelconque des revendications précédentes dans lequel l'oscillation a un déplacement de 5 à 50 mm.
 
5. Procédé selon l'une quelconque des revendications précédentes dans lequel l'échantillon de tissu est un rein, un muscle, un os, un tissu adipeux, un cartilage, un poumon, la vessie, la cornée, la peau, le foie, la rate, le placenta, l'intestin, le pancréas, la prostate, le sein ou le cœur.
 
6. Procédé selon l'une quelconque des revendications précédentes dans lequel l'agent osmotique est un agent hypotonique, facultativement de l'eau déminéralisée.
 
7. Procédé selon l'une quelconque des revendications précédentes dans lequel le détergent comprend l'éther p-(1,1,3,3-tétraméthylbutyl)-phénylique de polyéthylène glycol (Triton X100™), SDS et/ou SdC.
 
8. Procédé selon l'une quelconque des revendications précédentes dans lequel l'échantillon de tissu est traité avec le détergent en combinaison avec une protéase, facultativement la trypsine.
 
9. Procédé selon l'une quelconque des revendications précédentes dans lequel les cellules dans l'échantillon de tissu sont mécaniquement endommagées dans l'étape (i), facultativement par congélation et décongélation de l'échantillon de tissu.
 
10. Procédé selon l'une quelconque des revendications précédentes comprenant l'une quelconque de ;

A)

(a) l'exposition de l'échantillon à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 0 à 50 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes,

(e) la répétition de l'étape (d),

(f) l'exposition de l'échantillon à une solution saline,

(g) la répétition de l'étape (f) 0 à 10 fois,

(h) la répétition des étapes (a) à (e) 0 à 10 fois,

(i) l'exposition de l'échantillon à une solution saline, et

(j) la répétition de l'étape (i) 0 à 10 fois ; ou

B)

(a) l'exposition de l'échantillon de tissu à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 4 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes,

(e) la répétition de l'étape (d),

(f) l'exposition de l'échantillon à une solution saline,

(g) la répétition de l'étape (e) 4 fois,

(h) la répétition des étapes (a) à (e),

(i) l'exposition de l'échantillon à une solution saline, et

(j) la répétition de l'étape (i) 2 fois ; ou

C)

(a) l'exposition de l'échantillon de tissu à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 10 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes,

(e) la répétition de l'étape (d),

(f) l'exposition de l'échantillon à une solution saline,

(g) la répétition de l'étape (f) 4 fois,

(h) la répétition des étapes (a) à (e),

(i) l'exposition de l'échantillon à une solution saline, et

(j) la répétition de l'étape (i) 2 fois ; ou

D)

(a) l'exposition de l'échantillon à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 11 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes,

(e) la répétition de l'étape (d),

(e) l'exposition de l'échantillon à une solution saline pendant 2 minutes,

(f) l'exposition de l'échantillon à une solution saline pendant 5 minutes, et

(g) la répétition de l'étape (f) 2 fois ;

facultativement dans lequel l'échantillon de tissu est un échantillon de foie.


 
11. Procédé selon l'une quelconque des revendications 1 à 10 comprenant ;

(a) l'exposition de l'échantillon à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 5 à 10 fois,

(c) l'exposition de l'échantillon à un détergent,

(d) la répétition de l'étape (a) 5 à 10 fois,

(e) l'exposition de l'échantillon à une solution saline,

(f) facultativement la répétition des étapes (a) à (e) un ou plusieurs fois ;
facultativement dans lequel l'échantillon de tissu est un échantillon de rein ou de cœur.


 
12. Procédé selon la revendication 11 dans lequel l'échantillon de tissu est amené à osciller verticalement à 50 Hz.
 
13. Procédé selon l'une quelconque des revendications 1 à 10 comprenant l'une quelconque de ;

A)

(a) l'exposition de l'échantillon à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 19 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(e) la répétition de l'étape (d),

(f) l'exposition de l'échantillon à une solution saline

(g) la répétition de l'étape (f) 4 fois,

(h) la répétition des étapes (a) à (e) 2 fois,

(i) l'exposition de l'échantillon à une solution saline,

(j) la répétition de l'étape (i) 2 fois ; ou

B)

(a) l'exposition de l'échantillon à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 9 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes

(e) la répétition de l'étape (d)

(f) la répétition des étapes (c) à (e),

(g) l'exposition de l'échantillon à une solution saline,

(h) la répétition de l'étape (g) 2 fois ; ou

C)

(a) l'exposition de l'échantillon à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 10 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes,

(e) la répétition de l'étape (d),

(f) l'exposition de l'échantillon à une solution saline, et

(g) la répétition de l'étape (f) 2 fois ; ou

D)

(a) l'exposition de l'échantillon à de l'eau déminéralisée

(b) la répétition de l'étape (a) 20 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes

(e) la répétition de l'étape (d),

(f) l'exposition de l'échantillon à une solution saline,

(g) la répétition de l'étape (f) 5 fois,

(h) la répétition des étapes (a) à (d) 5 fois,

(i) l'exposition de l'échantillon à une solution saline, et

(j) la répétition de l'étape (i) 2 fois ; ou

E)

(a) l'exposition de l'échantillon à de l'eau déminéralisée,

(b) la répétition de l'étape (a) 4 fois,

(c) l'exposition de l'échantillon à un détergent et une protéase pendant 2 minutes,

(d) l'exposition de l'échantillon à un détergent et une protéase pendant 4 minutes,

(e) la répétition de l'étape (d),

(f) l'exposition de l'échantillon à l'acide peracétique (PAA),

(g) la répétition de l'étape (f) 1 fois,

(h) l'exposition de l'échantillon à l'hydroxyde d'ammonium (NH4OH),

(i) la répétition de l'étape (h) 2 fois,

(j) l'exposition de l'échantillon à de l'eau déminéralisée,

(k) la répétition de l'étape (j) 10 fois,

(l) la répétition des étapes (c) et (d),

(m) l'exposition de l'échantillon à une solution saline,

(n) la répétition de l'étape (m) 2 fois ;

facultativement dans lequel l'échantillon de tissu est un échantillon de foie, de pancréas ou d'intestin.


 
14. Procédé selon la revendication 13 dans lequel l'échantillon de tissu est amené à osciller horizontalement à 30 Hz.
 
15. Procédé selon l'une quelconque des revendications précédentes dans lequel l'échantillon de tissu est exposé à l'agent osmotique et/ou au détergent pendant 2 à 4 minutes dans chaque répétition des étapes (ii) et (iii).
 
16. Procédé selon l'une quelconque des revendications 1 à 15 comprenant

(i) la stérilisation de l'échafaudage après décellularisation et/ou

(ii) le repeuplement de l'échafaudage décellularisé avec des cellules pour produire un échafaudage recellularisé.


 
17. Procédé de modélisation de maladie comprenant ;
la production d'un échafaudage tissulaire décellularisé par un procédé selon l'une quelconque des revendications 1 à 15, facultativement le repeuplement de l'échantillon avec des cellules pour produire un tissu recellularisé, et,
la détermination de l'effet d'un composé, d'un médicament, d'un agent biologique, d'un dispositif ou d'une intervention thérapeutique sur l'échafaudage.
 
18. Procédé d'identification de biomarqueur de matrice extracellulaire (MEC) comprenant ;
la production d'un échafaudage tissulaire décellularisé par un procédé selon l'une quelconque des revendications 1 à 15,
l'exposition de l'échafaudage à une protéase, et
la détermination de la dégradation d'un ou plusieurs composants de l'échafaudage par la protéase, et
l'identification d'un ou plusieurs composants dégradés par la protéase en tant que biomarqueurs de MEC.
 
19. Procédé de diagnostic de la présence d'un tissu malade chez un individu comprenant ;
la production d'un échafaudage décellularisé par un procédé selon l'une quelconque des revendications 1 à 15 à partir d'un tissu de l'individu, et
la détermination de la structure et/ou la composition de l'échafaudage,
dans lequel une structure et/ou composition aberrante de l'échafaudage indique que le tissu est malade chez l'individu.
 




Drawing









































Cited references

REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description