CRYOPRESERVATION

Abstract

Methods and materials for the cryopreservation of cellularised scaffolds used for therapeutic or pharmacological testing purposes that provide a cultured scaffold on which cells have been seeded, equilibrate the cellularised scaffold with a cryopreservative composition comprising culture medium and between 5 and 30% of a cryoprotectant such as DMSO, freeze the equilibrated cellularised scaffold by reducing the temperature continuously by about 1 C./minute to about 80 C., and store the frozen cellularised scaffold at a temperature of between 135 C. and 198 C.

Claims

1. A method for the cryopreservation of a cellularised scaffold, which method comprises: (i) providing a cellularised scaffold; (ii) equilibrating said cellularised scaffold with a cryopreservative composition comprising culture medium and between 5 and 30% of a cryoprotectant; (iii) freezing the equilibrated cellularised scaffold by reducing the temperature at between 0.8 C. and 1.2 C./minute to between 78 C. to 82 C.; iv) storing the frozen cellularised scaffold at a temperature of between 135 C. and 198 C.

2. A method as claimed in claim 1 wherein step (iii) comprises freezing the equilibrated cellularised scaffold by continuously reducing the temperature at about 1 C./minute to about 80 C.

3. A method as claimed in claim 2 wherein step (i) comprises: (ia) providing an acellular scaffold; (ib) seeding the acellular scaffold with the cells; (ic) culturing the seeded scaffold to produce said cellularised scaffold.

4. A method as claimed in claim 3 wherein step (ia) comprises: (ia-1) providing a tissue or organ or sample thereof; (ia-2) decellularizing said tissue or organ or sample thereof using one or both of detergents and enzymes to provide an acellular scaffold.

5. A method as claimed in claim 1 wherein the scaffold is a sheet scaffold, which is preferably tubular.

6. A method as claimed in claim 1 wherein the scaffold is derived from an organ or tissue which has been decellularized, wherein the organ is preferably luminal.

7. A method as claimed in claim 1 wherein the scaffold is of non-human origin.

8. A method as claimed in claim 1 wherein the cellularised scaffold is a tissue engineered oesophagus construct, which is optionally suitable for a neonate or infant.

9. A method as claimed in claim 7 wherein the cellularised scaffold is derived from decellularised oesophagus seeded with mesoangioblasts and fibroblasts.

10. A method as claimed in claim 9 wherein the culture medium is FBS plus Megacell medium comprising 1% Penicillin Streptomycin; 1% L-Glutamine; 1% non-essential amino acids; 0.1 mM Beta Mercapto Ethanol and 5 ng/ml Basic FGF.

11. A method as claimed in claim 1 wherein the scaffold is spherical, cuboid, cylindrical, hexagonal prismatic, conical, frustoconical, or pyramidal.

12. A method as claimed in claim 11 wherein the scaffold is cuboid.

13. A method as claimed in claim 1 wherein the scaffold is derived from a solid organ which has been decellularized.

14. A method as claimed in claim 11 wherein the cellularised scaffold is tissue engineered liver.

15. A method as claimed in claim 14 wherein the cellularised scaffold is a decellularised liver tissue seeded with human hepatic cells, which are optionally HepG2 cells.

16. A method as claimed in claim 1 wherein the scaffold is a hydrogel scaffold.

17. A method as claimed in claim 16 wherein the cellularised scaffold is a hydrogel scaffold seeded with human hepatic cells, which are optionally HepG2 cells.

18. A method as claimed in claim 17 wherein the culture medium comprises FBS plus a liver-cell supporting medium.

19. A method as claimed in claim 14 wherein the cellularised scaffold is liver model tissue for pharmacological research.

20. A method as claimed in claim 1 wherein the cellularised scaffold is tissue engineered lung, intestine, pancreas, muscle or bladder.

21. A method as claimed in claim 1 wherein the cryopreservative composition comprises 80% or more culture medium.

22. A method as claimed in claim 21 wherein the cryopreservative composition comprises between 5% to 15%, more preferably 8 to 12%, more preferably about 10% cryoprotectant.

23. A method as claimed in claim 1 wherein the cryoprotectant is selected from the list consisting of: dimethyl sulfoxide (DMSO); Ethylene glycol; Glycerol; 2-Methyl-2,4-pentanediol; Propylene glycol; Sucrose; Trehalose.

24. A method as claimed in claim 1 wherein step (ii) is carried out below ambient temperature, optionally at about 0 to 4 C.

25. A method as claimed in claim 1 wherein step (iv) is carried out by placing the equilibrated cellularised scaffold within one or more containers in the vapour phase of liquid nitrogen such as to achieve a temperature of about 160 C.

26. A method as claimed in claim 1 wherein step (iv) is carried out for at least 1, 2, 4, or 4 weeks.

27. A method as claimed in claim 1 further comprising: (v) thawing the frozen cellularised scaffold rapidly in a water bath, optionally at 37 C.

28. A cryopreserved cellularised scaffold obtained according to the method of claim 1.

29. A thawed cryopreserved cellularised scaffold obtained according to the method of claim 27.

30. A kit comprising a cryopreserved cellularised scaffold of claim 28 and one or more containers.

31. A kit according to claim 30, wherein said kit further comprises instructions or labeling for the use of said kit for therapeutic purposes or for pharmacological research purposes.

32. A method of treatment of a subject with a chronic illness leading to organ failure, which method comprises: (i) providing a cellularised scaffold which is a tissue engineered organ replacement using autologous cells of the subject; (ii) cryopreserving the organ replacement according to the method of claim 1; (iii) thawing the organ replacement when it is required by said subject; (iv) treating said subject using said organ replacement.

33. A system for providing allogeneic tissue engineered organ replacements, which system comprises: (i) providing a plurality cellularised scaffolds which are allogeneic tissue engineered organ replacements using universal donor iPS cells; (ii) cryopreserving the organ replacements according to the method of claim 1; (iii) identifying a subject in need to an organ replacement; (iv) identifying a compatible cryopreserved allogeneic tissue engineered organ replacement; (v) thawing the organ replacement when it is required by said subject; (vi) treating said subject using said organ replacement.

34. A method for providing a three dimensional engineered micro-scaffold for use in pharmacological research purposes, which method comprises: (i) providing a cellularised scaffold which is a three dimensional engineered micro-scaffold suitable for pharmacological research purposes; (ii) cryopreserving the engineered micro-scaffold according to the method of claim 1; (iii) thawing the micro-scaffold when it is required.

35. A cellularised scaffold or cryopreserved cellularised scaffold for use in the method or system of claim 32.

36. Use of a cellularised scaffold or cryopreserved cellularised scaffold for the preparation of a medical implant or material, for use in a method or system of claim 32.

Description

FIGURES

[0146] FIG. 1-FIG. 2C: Cryopreservation of a recellularised tissue engineered oesophagus.

[0147] Rat decellularised oesophagi were seeded with Luc.sup.+Zs-Green.sup.+ human mesoangioblasts and mouse fibroblasts and mouse GFP.sup.+ neural crest cells, cultured in a bioreactor for up to 11 days then cryopreserved with the developed protocol for two weeks, thawed with developed protocol and cultured for a further 14 days.

[0148] The cells were tracked using bioluminescence imaging with an In Vivo Imagine System (IVIS, Perkin Elmer) pre and post cryopreservation. H&E staining shows the cells in the scaffold 14 days post-cryo (FIG. 2A, FIG. 2B). In FIG. 2B, the results for the MABs are shown on the left and the MABs+FBs on the right for each time point.

[0149] In FIG. 2C a representative section of the cryo-preserved oesophagus containing human mesangioblasts and fibroblasts and mouse GFP+ neurocrest cells.

[0150] FIG. 3. Cryopreservation of recellularised human liver scaffolds and liver hydrogel.

[0151] 0.5M HepG2 were seeded on 16 hydrogels and 16 HL-68. Half of each scaffold type were cryopreserved for 2 weeks using developed method. Post-cryopreservation, there was no significant different between the fresh scaffolds and the thawed cryopreserved scaffolds. In the histogram, the results for HL-68 are shown on the left and the results for hydrogel shown on the right for each time point.

[0152] FIG. 4A-FIG. 4C: Maintenance of cell viability after storage

[0153] Oesophageal scaffolds were seeded with Luc.sup.+ZsGreen.sup.+hMAB+mFB and were cultured in static conditions and then cryopreserved for 2 weeks. The results demonstrate that the bio-engineered muscle cryopreserved with a slow-cooling process show maintenance of cell viability after storage.

[0154] In FIG. 4A bioluminescence images of a representative scaffold seeded with Luc+ZsGreen+hMAB+mFB and cultured in static for 8 days (pre-cryo) and for further 7 days after 2 weeks of cryopreservation are shown. Locations of detected bioluminescence radiance are indicated with an arrow.

[0155] In FIG. 4B a representative image of MTT colorimetric assay performed on a seeded-scaffold following cryopreservation. Scale bar: 1 mm.

[0156] FIG. 4C shows average radiance detected using bioluminescence imaging before and after cryopreservation at different time points. Data: meanSEM (n=3).

EXAMPLES

Example 1Materials and Methods

[0157] Confirming Scaffold Integrity

[0158] Histology

[0159] Samples are fixed for 24 hours in 10% neutral buffered formalin solution in PBS (pH 7.4; Sigma, UK) at RT, washed in dH.sub.2O, dehydrated in graded alcohol, embedded in paraffin and sectioned at 5 m. Tissue slides are stained with haematoxylin and eosin (H&E; Leica, Germany).

[0160] DNA Quantification

[0161] DNA is isolated using a tissue DNA isolation kit following the manufacturer's instructions (PureLink Genomic DNA MiniKit, Invitrogen, UK).

[0162] ECM Component Quantification

[0163] Collagen, elastin and glycosaminoglycan (GAG) content can be quantified using the total collagen assay kit (Biocolor, UK), the FASTIN elastin assay and the GAG assay kit (Biocolor, UK) respectivelysee [14]

[0164] Biomechanical Testing

[0165] To evaluate the biomechanical properties of oesophagi, specimens can be tested and subjected to uniaxial longitudinal tension until failure [12]. Uniaxial tension may be applied using an Instron 5565, with specimens in the form of flat dumbbells (20 mm) loaded at a constant tension rate of 100 mm/min. The thickness of the samples can be measured using a digital electronic micrometer (RS components, US) at three places of the dumbbell and averaged.

[0166] Scanning Electron Microscopy (SEM)

[0167] Samples are fixed in 2.5% glutaraldehyde (Sigma, UK) in 0.1 M phosphate buffer and left for 24 hrs at 4 C. SEM may be performed as described in [14].

[0168] Confirming Cell Viability, Number and Metabolic Activity

[0169] Cell viability may be carried out according to methods well known in the art, for example as described in U.S. Pat. No. 6,638,709. These include assessing construct cell density, the total number of viable cells per unit area; cell viability, the percent of the total number of cells that are viable; and metabolic activity, a measure of the overall vigor of the viable cells in terms of their ability to metabolize nutrients and perform other cell maintenance functions. Additional measurements include histologic examination of the cellularised construct for the presence, configuration, and distribution of cells within and on the construct.

[0170] Briefly, cell number and cell viability can be measured by releasing cells from the construct and determining cell viability and cell number by a Hemocytometer using Trypan Blue dye exclusion to differentiate living from dead cells.

[0171] Metabolic Activity may be measured using samples incubated with Alamar Blue dye. The assay measures mitochondrial activity using a non-cytotoxic Alamar Blue dye which diffuses into the cell mitochondria and undergoes a reduction-oxidation reaction to give a fluorescent product that is read by a fluorescent spectrophotometer. Metabolic Activity may be measured using an MTT assay. The yellow MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically active cells by the action of dehydrogenase enzymes to generate reducing equivalents such as NADH and NADPH, resulting in a purple formazan that can be solubilised and quantified by spectrophotometric means. In this way, the MTT assay can be used to measure cell viability.

[0172] Cell viability can also be measured using an assay that detects markers of apoptosis, for example, a caspase-3 assay. An exemplary caspase-3 assay is described in the examples.

[0173] Histology requires a visual assessment of the structure and morphology of the construct and cells therein (see Examples herein).

[0174] IVIS Imaging:

[0175] Lentivirus Production

[0176] The lentiviral transfer vector pHIV-LUC-ZsGreen (used in FIG. 2A) was a gift from Dr. Bryan Welm (Department of Surgery, University of Utah, purchased through Addgene Inc. MA, USA, Plasmid #39196) and was used to generate a lentivirus containing both ZsGreen florescent protein and firefly luciferase from an EF1-alpha promoter. This third generation lentivirus required the packaging plasmids pRSV-Rev (Addgene Plasmid #12253) and pMDLg/pRRE (Addgene #12251) as well as the VSV-G envelope plasmid pMD2.G. (Addgene Plasmid #12259).

[0177] Briefly, lentiviral vectors were produced by co-transfecting 293T cells with the above plasmids. Transfection of plasmids was in 293T cells using a jetPEI/plasmid mix according to manufacturer's instructions. After 6 hours at 37 C., the medium (DMEM containing 10% FBS; Gibco, U.K.) was exchanged for virus collection. After 24 hours, this virus-containing medium was purified by centrifugation at 2500 rpm (4 C.) and filtered through a 0.45 m membrane. Medium was ultracentrifuged at 50,000 g for 2 hours at 4 C. (SW28 rotor, Optima LE80K Ultracentrifuge, Beckham, High Wycombe, UK). The viral pellet was re-suspended in 100 l pre-cooled serum-free DMEM (Gibco, U.K.) and the virus was stored at 80 C. until use.

[0178] Viral titres were calculated by transduction efficacy in HeLa cells, a cell line known to be permissive to viral transduction. HeLa cells were expanded in complete DMEM. Cells were seeded at 5104 cells per well in a 24-well plate. A dilution series (1:5) from 20 l/ml virus to 0.0032 l/ml virus was created in a total volume of 500 l per well. Cells were cultured overnight and changed for fresh medium the following day. Transduction efficacy was determined by flow cytometric analysis of the proportion of cells expressing the fluorescent protein ZsGreen 72 hours after transfection. Viral titres were calculated with the following formula.

Viral titre (iu/ml)=Number of cells seededpercentage of florescent positive cells/Volume of virus (ml)

[0179] Viral titres were calculated from volume of virus used to transduce cells at 15-25% transduction efficacy.

[0180] Lentiviral Transduction of Stromal Cells and FACS Sorting.

[0181] Stromal cells derived from muscle were transduced with the lentivirus as described above but scaled to T25 flasks and tested at increasing MOI. Transduction efficacy was determined by FACs as a percentage of cells transduced. In order to obtain a pure population of transduced cells, cells were FACS sorted following expansion of cells by one passage. Briefly cells were trypsinized, centrifuged and 1106 cells re-suspended in 500 l of FACS buffer and sorted using a FACSAria (BD Biosciences). Sorted cells were expanded by a further passage and checked by flow cytometry to ensure a pure population of transduced cells were maintained and used for downstream experiments.

[0182] Bioluminescent Imaging in a Bioreactor

[0183] Culture medium containing 150 g/ml D-Luciferin was injected into the internal chamber of the bioreactor via the 3-way luer taps and imaged as described above. The bioreactor was placed on the stage and imaged. Stage D was used for zoomed out images of the entire reactor and stage C for all other images and analysis.

[0184] Other Materials

[0185] Megacell medium comprises 5% FBS, 1% Penicillin Streptomycin, 1% L-Glutamine, 1% non-essential amino acids, 0.1 mM Beta Mercapto Ethanol and 5 ng/ml Basic FGF.

[0186] For cryopreservation the media composition was 50% Fetal Bovine Serum (FBS), 40% MEGACELL Medium with supplements (described above), and 10% dimethyl sulfoxide (DMSO, Me2SO; Sigma, UK).

[0187] Slow cooling was achieved using Mr Frosty (Nalgene) freezing containers. Nalgene freezing containers were kept at 80 C. overnight.

Example 2Oesophagus

[0188] Rat decellularized oesophagi seeded with human mesoangioblasts (MABs), mouse fibroblasts (FBs) and mouse neural crest cells, were cultured in a bioreactor for up to 11 days and then frozen with the following protocol: [0189] The seeded scaffold (size 7+20 mm length) was placed in a cryovial (size: 2 mL) with 500 L FBS. [0190] The vial was kept in ice throughout the process. [0191] Another 500 L were added of a solution of Megacell medium containing 20% DMSO. [0192] The vial was transferred in a Nalgene freezing container and kept at 80 C. overnight. [0193] Samples were then placed and stored in the vapour phase of liquid nitrogen at approximately 160 C. [0194] After 2 to 4 weeks in the liquid nitrogen container, vials were rapidly thawed at 37 C. and samples transferred in 10-20 mL culture medium (Megacell supplemented with FBS and antibiotic) at 37 C. under mild agitation for 20 minutes. [0195] Samples were then transferred to a culture petri dish with fresh culture medium and left in static culture for up to 7 days.

[0196] Cell viability and localization were confirmed with bioluminescence and histology. Scaffolds seeded with either MABs alone or co-seeded with MABs+FBs showed comparable survival after freezing, measuring radiance with IVIS before and after cryopreservation (FIG. 2A, FIG. 2B). Cell growth was clear immediately after thawing, with a continuous increase in bioluminescence emitted by seeded scaffolds throughout the 14 days of culture post-cryopreservation. At the end of the culture, scaffolds were analysed with histology to confirm cellular presence and in the correct localization of the oesophageal muscle layer. These results indicated not only cell survival after cryopreservation independently by the type of cell seeded, but also highlighted their capability to grow perfectly after thawing.

[0197] Cell differentiation and orientation within the scaffold was further assessed with immunostaining for hNuclei and GFP (FIG. 2C). Scaffolds co-seeded with a combination of MABs, FBs and GFP+ neural crest cells were cultured in dynamic conditions for 11 days, cryopreserved for 14 days, thawed and cultured for further 7 days. Immunofluorescence imaging showed how cell orientation, GFP+ neural crest cell morphology and cell-cell interaction were preserved after cryo-preservation, with comparable results to non-stored scaffolds.

Example 3Liver

[0198] Human decellularized liver cubes (555 mm) or human decellularized liver-derived Hydrogel cubes (555 mm) seeded with HepG2 cell line, cultured in static conditions for up to 10 days were frozen with the following protocol: [0199] The seeded scaffold was placed in a cryovial (size: 2 mL) with 500 L FBS. [0200] The vial was kept in ice throughout the process. [0201] Another 500 L were added of a solution of alpha MEM containing 20% DMSO. [0202] The vial was transferred in a Nalgene freezing container and kept at 80 C. overnight. [0203] Samples were then placed and stored in the vapour phase of liquid nitrogen at approximately 160 C. [0204] After 2 weeks in the liquid nitrogen container, vials were rapidly thaw at 37 C. and samples transferred in 5-10 mL culture medium (alpha MEM containing 10% FBS, 1% Antibiotic, 1% 1 mM sodium pyruvate, 1% non-essential AA solution 100)) at 37 C. under mild agitation for 20 minutes. [0205] Samples were then transferred to a culture petri dish with fresh culture medium and left in static culture for 3 days.

[0206] Subsequent analysis showed that the cells survived freezing and showed albumin production comparable with pre-freezing samples.

[0207] Albumin measurement: was performed using Abcam's Serum Albumin (ALB) in vitro SimpleStep ELISA (Enzyme-Linked Immunosorbent Assay) kit. This is designed for the quantitative measurement of Serum Albumin protein in human serum and plasma.

[0208] The SimpleStep ELISA employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

Example 4Maintenance of Cell Viability after Storage

[0209] An experiment was designed to further demonstrate maintenance of cell viability following storage of a scaffold prepared and cryopreserved using the developed protocol.

[0210] Oesophageal scaffolds seeded with Luc.sup.+ZsGreen.sup.+MAB+FB were cultured in static conditions and then cryopreserved for 2 weeks in the same manner as described for Example 2. After storage, the samples were thawed and grown for 7 days in static culture. Cell viability was detected at various stages pre- and post-cryopreservation using bioluminescence (FIG. 4A, FIG. 4C) and an MTT assay (FIG. 4B).

[0211] After 7 days of culture the number of caspase 3 positive (caspase3.sup.+) cells was determined using immunofluorescence. Tissue samples were fixed in paraformaldehyde and frozen. 7-10 m thick sections were cut with a cryostat and incubated with primary and secondary antibodies diluted in 1% Goat Serum/PBS/0.01% Triton X-100. Images were acquired with a Zeiss LSM 710 confocal microscope (Zeiss) and processed using ImageJ and Adobe Photoshop. Manual cell counting was performed to calculate the number of caspase3.sup.+ cells over the total number of DAPI.sup.+ cells in random sections from different regions of scaffolds.

[0212] The results demonstrate that the bio-engineered muscle that were cryopreserved with a slow-cooling process showed maintenance of cell viability after storage. Post-thawing, scaffolds showed a slight reduction in cell viability when compared to before cryopreservation. However, cells were able to recover and grow for up to 7 days in static culture, as confirmed by bioluminescence reading and MTT assay (FIG. 4A-FIG. 4C). Very few caspase3.sup.+ cells (<0.1%) were found in the cryopreserved scaffolds after 7 days of culture (data not shown).

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