Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
  • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution

Definitions

• the present invention relates to a method for producing a decellularized adipose tissue matrix, to the decellularized adipose tissue matrix obtainable by it and to its uses.
• the adipose tissue obtainable by the method has multiple applications in tissue engineering and regenerative therapy, especially in the topical treatment of wounds.
• Skin ulcers are open lesions that usually involve the destruction of the epidermis and the dermis. In the worst of cases, the deep lesions also involve the destruction of the hypodermis, the lower layers of the skin. When the ulcers take more than 6 weeks to scar, they are classified as chronic. Chronic ulcers can be caused by a variety of different pathologies ranging from infections and local ischemia to cardiovascular dysfunctions. These have a high prevalence, inflict a heavy burden on the health systems worldwide and in many cases are not managed easily in the clinic.
• acellular biological matrices are products that are derived from animal tissue, and which can be applied topically at the site of the wound to stimulate and replace the disrupted or missing extracellular matrix (ECM). When applied topically, they provide a scaffold rich in proteins, glycolipids and other ECM components to which cells migrate and proliferate, enabling tissue regeneration.
• Biological matrices are currently being explored in several applications, not only in regenerative therapy but also for tissue engineering such as internal implants and orthopedic devices. In particular, an interesting application, unrelated to therapy, is the use of such matrices for in vitro culture devices.
• acellular matrices resemble an actual tissue and therefore can be aptly used in any cell-culture device to promote the maintenance and growth of a cell culture.
• matrices that are capable of regenerating the hypodermis are still lacking, both because of their sub-optimal properties and because they are not obtained from subcutaneous adipose tissue.
• any method for processing an initial tissue to yield an acellular matrix must ensure that the final product is endowed with an overall structure and mechanical properties that resemble that of the native ECM matrix, it must also preserve the proteins and other macromolecular components that enable the matrix to be used as a scaffolding for the regenerative processes to take place, and most crucially must be devoid of any nuclear components such as nuclear debris and DNA that would render it pro-inflammatory and immunogenic.
• any method of acellular matrix production must make sure that the series of treatments applied maximize the elimination of the immunogenic components of the original tissue and at the same time minimize the loss of structural
• the chemical treatments fall into different categories such as detergents, organic solvents, acidic and alkaline solutions, etc. As it is disclosed in this reference, it is extremely rare to only rely in a single chemical treatment to decellularize a tissue, and it is generally regarded as more advantageous to combine numerous chemicals and biochemicals in a series of short washes to ensure the efficiency of the treatments and hence a proper decellularization. However, the use of chemicals implies a risk in terms of contamination with undesirable residues which are not apt for
• extracellular matrix produced from adipose tissue and methods for producing it, which can be composed of a series of treatments including delipidation with lipases, protein digestion with a range of proteases such as pepsin, papain, matrix metal loproteases (MMPs) and trypsin, nucleic acid digestion with endonucleases, exonucleases, DNAses and RNAses.
• this method is characterized by a step where detergents such as sodium dioxycholate, sodium docecyl sulphate or Triton X-100 are used.
• US201 1/015101 1 discloses a method for decellularizing adipose tissue comprising subjecting the adipose tissue to a series of enzymatic digestions and a series of solvent extractions so that the final acellular adipose tissue has a well preserved 3D structure for regenerative therapy.
• the methods described can involve protocols where a series of treatments can be combined, including treatments with proteases, DNAse and RNAse, chelating agents such as EDTA, detergents such as Triton-X100 and lipases.
• US2013/0202563 discloses a method of producing a cell growth scaffold from adipose tissue comprising a washing step with n-propanol, isopropanol or a mixture thereof.
• the treatment does not imply the use of enzymes for the degradation of the original tissue components, and is mostly based on the use of alcohols.
• WO201 1/132089 discloses a method for decellularizing tissues which comprises contacting the original tissue with different chemical solutions and surfactants and then treating the resulting surfactant-treated tissues
• the method of the invention comprises a reduced number of steps that render it substantially simpler than many methods found in the art, thanks to the particular conditions that have been found to be optimal in terms of performance.
• the method involves the use of only two biochemical (enzymatic) treatment steps, namely lipoprotein lipase and a nuclease of high (biopharmaceutical) quality.
• the method does not involve a series of lengthy, serial chemical treatments, which ensures that it can be applied without raising any concerns related to the safety of the final decellularized matrix.
• the two enzymatic treatments are carried out under very specific conditions of concentration, duration and temperature, and only with enzymes of biopharmaceutical quality, which guarantee the quality of the decellularized matrix obtained.
• the method will usually entail the use of resected tissue (although it could be applied to lipoaspirates as well), it does not feature a combination of thorough chemical treatments and does not involve the use of reagents of animal origin, organic solvents or any aggressive mechanical treatments.
• a first aspect of the present invention is a method of decellularizing adipose tissue comprising the steps of: a) Laminating the adipose tissue; b) Treating the adipose tissue resulting from step a) with a lipoprotein lipase at 32-42 degrees Celsius and at a concentration of 10-55u/100mg; and c) Treating the adipose tissue resulting from step b) with a nuclease, at 32-42 degrees Celsius and at a concentration of 709-1433u/mg; for the necessary period of time for the adipose tissue to have a total of DNA content equal to or less than 50 ng/mg.
• the DNA content is referred to the ng of DNA per mg of dry weigth decellularized human adipose tissue, as described in the examples.
• the decellularized matrix obtained by the method is endowed with properties expected for therapeutic applications as it has a very low DNA content (usually far below 50ng/mg, which is the threshold above which in vivo immune reactions have been described according to Crapo, PM., et. al. "An overview of tissue and whole organ decellularization processes" Biomaterials 201 1 , vol. 32, pp. 3233-3243), a desirable percentage of extracellular matrix protein (in particular perlecan, elastin, collagen type I and IV) and a
• the matrix has a global structure resembling that of the original tissue, which maximizes the likelihood of success in regenerative medicine. Therefore, inventors have found a method that neatly strikes a balance between elimination of unwanted components such as nuclear debris and other immunogenic DNA and preservation of structural integrity of the original tissue.
• the method can be applied with subtle variations that allow obtaining a decellularized matrix with the desired qualities but with varying degrees of triglyceride content, which is an advantage since depending on the final application the matrix might need a higher or lower content in lipids.
• a second aspect of the present invention is a decellularized adipose tissue matrix obtainable by the method of the first aspect of the invention.
• the matrix obtainable by the method can find a myriad of different applications, including the most restrictive and demanding ones such as the healing of deep wounds and ulcers that affect the
• the matrix obtainable by the first aspect is characterized by an absence of organic solvents, low residual content in terms of immunogenic components ensuring a minimal host response when applied in therapy, remarkable structural and functional similarities with the endogenous tissue, rich in basement membrane proteins and preserved overall architecture.
• a third aspect of the present invention is a powder, foam, particle or hydrogel comprising the decellularized adipose tissue according to the second aspect of the invention.
• a fourth aspect of the invention is a biocompatible scaffold or biocompatible coating comprising the decellularized adipose tissue according to the second aspect of the invention or the powder, foam, particle or hydrogel of the third aspect of the invention.
• a fifth aspect of the invention is the decellularized adipose tissue according to the second aspect of the invention, the powder, foam, particle or hydrogel according to the third aspect of the invention, or the biocompatible scaffold or biocompatible coating according to the fourth aspect of the invention for use in tissue engineering and regenerative therapy.
• FIG. 1 H&E stained sections of normal and decellularized hAT (hAT and dhAT-LM4). Adipocyte (column a), fibrillary regions (column b, asterisk) and vascular structures (column c, arrows) are shown. Scale bar represents 200 ⁇ .
• FIG. 2 IHQ analysis of Collagen type I (fibrillary region, column a), collagen type IV (adipocyte region, column b), collagen type IV (vascular structures, column c), laminin (column d) and HSPG2 (column e) of normal and decellularized hAT (hAT and dhAT-LM4). Vascular structures have been identified (arrow). Scale bar represents 200 ⁇ .
• FIG. 4 H&E staining and IHQ analysis of collagen type-l of dhATs treated and untreated with the high quality nuclease Benzonase (LM3 and M8
• Scale bar represents 200 ⁇ .
• FIG. 5 H&E staining and IHQ analysis of collagen type I (a), collagen type IV (b), laminin (c) and HSPG2 (d) of dhATs untreated with trypsin/triton-x100 (M7).
• Adipocyte (I), fibrillary regions (II, asterisk) and vascular structures (III, arrow) are shown.
• LM3 dhAT For comparative purposes with treated dhAT see Figure 3 (LM3 dhAT). Scale bar represents 200 ⁇ .
• FIG. 6 Macroscopic images and relative triglyceride content (%) of original hAT and dhATs obtained by different Lipase incubation conditions in the decellularization process (LM1 -LM5, conditions described in Table 1 ). Results are shown as relative triglyceride content (%) considering original hAT as a 100%.
• FIG. 7 Macroscopic images and relative triglyceride content (%) of original hAT and dhATs obtained by the decellularization process with the lack of trypsin/triton-x100 pretreatment (M7) or Benzonase treatment (LM8). Results are shown as relative triglyceride content (%) considering original hAT as a 100%.
• FIG. 8 Macroscopic and microscopic images of the processed dhAT of the invention: a) macroscopic image of a powder obtained by micron ization, b) microscopic image of powder obtained by Scanning Electron Microscopy. Scale bar 100 ⁇ , c) macroscopic image of a porous scaffold obtained by freeze drying, d) microscopic image of a porous scaffold obtained by Scanning Electron Microscopy, e) macroscopic image of the application of plastic compression, f) macroscopic image of a sheet obtained by plastic
• decellularization refers to a process by which a tissue is submitted to one or more treatments in order to maximize the removal of cells present in it, leaving only the extracellular matrix (ECM) that is rich in structural proteins such as collagens, elastin, growth factors, and glycolipids.
• ECM extracellular matrix
• biocompatible scaffold refers to a substance with sufficient structural stability to provide a substrate to support, foster and promote the growth of living cells which make up a tissue. Such a scaffold can be used for recovery of a damaged tissue. The scaffold fills in the gap left by a wound, giving a structure to be colonized by cells and new blood vessels, ultimately leading to tissue regeneration.
• a first aspect of the present invention is a method of decellularizing adipose tissue comprising the steps of: a) Laminating the adipose tissue; b) Treating the adipose tissue resulting from step a) with a lipoprotein lipase at 32-42 degrees Celsius and at a concentration of 10- 55u/100mg; and c) Treating the adipose tissue resulting from step b) with a nuclease, at 32-42 degrees Celsius and at a concentration of 709-1433u/mg; for the necessary period of time for the adipose tissue to have a total of DNA content equal to or less than 50 ng/mg;
• the method of decellularizing adipose tissue is a method wherein the lipase treatment b) is carried out either: at a concentration of 10-30u/100mg for 39-49 hours at 32- 42 degrees Celsius or at
• the method of decellularizing adipose tissue is a method wherein in step b) the treatment is at a concentration of 25u/1 OOmg for 44 hours at 37 degrees Celsius or at a concentration of 50u/100mg for 23 hours at 37 degrees
• step c) is carried out either at a concentration of 709-719u/mg for 67-77 hours at 32-42 degrees Celsius or at a concentration of 1423-1433u/mg for 35-45 hours at 32-42 degrees Celsius or at a concentration of 1423- 1433u/mg for 67-77 hours at 32-42 degrees Celsius.
• the method of decellularizing adipose tissue is a method wherein step b) is carried out either at a concentration of 10-30u/100mg for 39-49 hours at 32-42 degrees Celsius or at a concentration of 45-55u/1 OOmg for 18-28 hours a 32- 42 degrees Celsius, and in step c) the treatment is at a concentration of 714u/mg for 72 hours at 37 degrees Celsius or at a concentration of 1428u/mg for 40 hours at 37 degrees Celsius or at a concentration of 1428u/mg for 72 hours at 32-42 degrees Celsius.
• the method of decellularizing adipose tissue is a method wherein in step b) the treatment is at a concentration of 25u/100mg for 4 hours at 37 degrees Celsius.
• the method of decellularizing adipose tissue is a method wherein in step b) the treatment is at a concentration of 50u/100mg for 23 hours at 37 degrees Celsius.
• the method of decellularizing adipose tissue is a method wherein in step c) the treatment is at a concentration of 714u/mg for 72 hours at 37 degrees Celsius. In another particular embodiment of the first aspect of the invention, the method of decellularizing adipose tissue is a method wherein in step c) the treatment is at a concentration of 1428u/mg for 40 hours at 37 degrees Celsius.
• the method of decellularizing adipose tissue is a method wherein in step c) the treatment is at a concentration of 1428u/mg for 72 hours at 37 degrees Celsius.
• the method of decellularizing adipose tissue further comprises a step between steps a) and b), wherein the adipose tissue resulting from step a) is treated with trypsin and triton-X100.
• this last step is characterized by the fact that the trypsin-tritonXI 00 treatment further comprises EDTA and it is carried out at 37 degrees Celsius overnight and Triton-X100 is at 1 % in v/v (1 ml/100ml).
• the method further comprises a step d) comprising freezing or liofiphilizing and sterilizing the adipose tissue resulting from step c).
• the sterilization is carried out with ethylene dichloride. In another particular embodiment the sterilization is carried out with ultraviolet light. In another particular embodiment the method is carried out under aseptic conditions.
• the method of decellularizing adipose tissue is a method wherein in steps b) and c) the treatments are treatments under vacuum and with stirring at 100-150 rpm.
• the stirring at 100-150 rpm is orbital stirring.
• the method of decellulanzing adipose tissue is a method wherein after steps a), b) and c), there is a washing step with buffer at room temperature, under vacuum and under stirring at 100-150 rpm, and wherein the washing step after step a) the buffer further comprises at least one antibiotic, at least one antimycotic and at least one protease inhibitor, in the washing step after step b) the buffer comprises at least one antibiotic, at least one antimycotic, at least one protease inhibitor and at least one lipase inhibitor and wherein the washing step after step c) the buffer further comprises at least one antibiotic, at least one antimycotic ,at least one protease inhibitor and at least one nuclease inhibitor.
• the method of decellulanzing adipose tissue is a method wherein in step b), the lipoprotein lipase treatment comprises Triton-X100 at 0.5% and a cofactor in phosphate buffer.
• the method of decellulanzing adipose tissue is a method wherein in step b), the lipoprotein lipase treatment comprises Triton-X100 at 0.1 % and a cofactor in phosphate buffer.
• steps b) and c) are carried out in the presence of a cofactor.
• the cofactor is magnesium (Mg+2).
• the method of decellulanzing adipose tissue is a method wherein in step a), the lamination of the adipose tissue is manual.
• the method of decellulanzing adipose tissue is a method wherein in step a), the laminated block sizes are 1 -15 cm long, 0.5-8 cm wide and 0.3-2 cm thick.
• the method of decellulanzing adipose tissue is a method wherein in step b), the lipoprotein lipase is selected from the group consisiting of lipase and phospholipase from bacterial origin (genus Pseudomonas, Staphyloccus, Bacillus, etc.), yeast origin (genus Candida aldicans, Candida Antarctica, Candida rugosa, Geotrichum asteroids, Geotrichumcandidium,, Trichosporonfermentans, Saccharomycopsis lipolytica, Yarrowia lipolytica), fungal origin (genus Penicillunn, Rhizopus, Rhizomucor, etc), and mammalian origin (porcine, bovine, horse, human, etc.).
• bacterial origin gene Pseudomonas, Staphyloccus, Bacillus, etc.
• yeast origin genus Candida aldicans, Candida Antarctica, Candida rugosa, Geotrichum asteroids, Geotrichum
• lipoprotein lipase acylglicerol lipase, triacylglicerol lipase, hormone sensitive lipase, pancreatic lipase, bile salt activated lipase, pancreatic lipase related protein 1 , pancreatic lipase related protein 2, phospholipase A1 , phospholipase A2, calcium independent phospholipase A2, endothelial lipase, phosphatidylinositol phospholipase A2, endogenous phospholipase C, phosphoinosositide phospholipase C, phospholipase C, lysophospholipase D and phospholipase D.
• the method of decellularizing adipose tissue is a method wherein in step c), the nuclease is Benzonase®.
• Benzonase is an endonuclease with very high pharmaceutical quality provided by Merck-Millipore, which states in its technical specifications:
• Benzonase® is a unique, genetically-engineered endonuclease that is only available from Merck Millipore. Produced in E.coli, this non-specific, recombinant endonuclease cleaves all kinds of DNA and RNA variants into fragments that comprise ⁇ 8 soluble base pairs. This leads to an utmost minimum of nucleic acid load.
• the method of decellularizing adipose tissue is a method wherein in step c), the nuclease is selected from the group consisiting of nucleases from bacterial origin (Serratia marcescens, Clostridium), yeast origin, fungal origin
• the nuclease is selected from the group consisiting of nucleases from bacterial origin (Serratia marcescens, Clostridium), yeast origin, fungal origin
• DNases deoxyribonuclases
• RNases ribonucleases
• DNase I DNase II
• DNase V micrococcal nuclease
• type I site-especific DNase RNase I
• RNase H RNase H
• RNase III RNase III
• RNase L RNase L
• RNase P nuclease S1
• yeast RNase RNase U2
• RNase T2 RNase T1
• RNase P4 RNase M5, RNase IX
• RNase E RNase E
• RNase D RNase alpha
• RNase PH RNase PH
• RNase AS RNase Phyb
• RNase J1 Pancreatic RNase, nanoRNase.
• a second aspect of the present invention is a
• the adipose tissue to be treated is of subcutaneous origin, such as mammary or
• the adipose tissue to be treated is obtained from lipoaspirate.
• the adipose tissue to be treated is obtained from resection.
• the adipose tissue derives from the epiplon or the omentum.
• the adipose tissue is mammal adipose tissue.
• the adipose tissue is human adipose tissue.
• the adipose tissue obtainable by the method of the first aspect has a total DNA content of 0.015-0.306 ng/mg. This range of DNA content is considered as very acceptable by the inventors as it is far below the 50ng/mg threshold described above.
• the first aspect of the invention allows obtaining a decellularized matrix that, although it is characterized by a highly preserved morphology when compared to the original tissue, it has a nuclear residual content which minimizes the likelihood of in vivo adverse immune reactions.
• the adipose tissue matrix further comprises foreign bioactive molecules.
• the natural or synthetic bioactive molecules are selected from the group consisting of growth factors, hormones, vitamins, antioxidants, anti-inflammatories, antibacterial, antifungal, wound healing accelerators/healing promoting agents, or mixtures thereof.
• the adipose tissue matrix further comprises i) synthetic polymers (PEG, poly(a-hydroxy esters), polystyrene, polyurethane and copolymers and blends) and ii) natural polymers (protein-based elastin, collagen, fibrin; or polysaccharide-based collagen, chitosan, hyaluronic acid, beta-glucans, gelatin, micro- and nano- cellulose and their derivatives).
• synthetic polymers PEG, poly(a-hydroxy esters), polystyrene, polyurethane and copolymers and blends
• natural polymers protein-based elastin, collagen, fibrin; or polysaccharide-based collagen, chitosan, hyaluronic acid, beta-glucans, gelatin, micro- and nano- cellulose and their derivatives.
• the adipose tissue matrix further comprises human or animal live cells (inside or on top of the material): somatic cells (adipocyte, fibroblast, osteocyte, osteoblast chondrocyte, chondroblast, myo-epithelium, bone marrow, macrophages, etc.), endothelial, stem cells or induced pluripotent stem cells.
• somatic cells adipocyte, fibroblast, osteocyte, osteoblast chondrocyte, chondroblast, myo-epithelium, bone marrow, macrophages, etc.
• endothelial stem cells or induced pluripotent stem cells.
• the decellularized adipose tissue has a total protein content of 100-931 pg/mg and a triglyceride content of 25-65% of the original tissue (w/w).
• the second aspect of the invention can be further processed by micronization, plastic compression, dissolution, freeze drying, self-assembling, chemical crosslinking and/or induced physical interactions, electrospinning, spin coating or 3D printing.
• a third aspect of the present invention is a powder, foam, particle or hydrogel comprising the decellularized adipose tissue according to the second aspect of the invention.
• the powder, foam, particle or hydrogel comprising the decellularized adipose tissue is mixed with further bioactive components such as growth factors, foreign structural proteins or scarring agents.
• the resulting composite mixture has additional advantages in terms of tissue regeneration.
• a fifth aspect of the invention is the decellularized adipose tissue according to the second aspect of the invention, the powder, foam, particle or hydrogel according to the third aspect of the invention, or the biocompatible scaffold or biocompatible coating according to the fourth aspect of the invention for use in tissue engineering and regenerative therapy
• the use is in combination with a matrix selected from the group consisting of Matriderm, Primatrix, Integra, Apligraf, Epicel and Graftjacket.
• the use is in combination with any other matrix available in regenerative medicine.
• the therapy is a therapy of wound healing.
• the manifold applications of the first aspect of the invention comprise: wound healing, tissue engineering, regenerative medicine, additive therapies, and cell carriers for the treatment of disease or damaged tissue and organs, healing or prevention of disease and restoration, correction or alteration of physiological functions of: i) connective tissue (adipose, skin, blood vessel, cartilage, bone etc.), ii) epithelial and endothelial tissue
• the wound healing is wound healing of deep wounds (type III and IV) involving the hypodermis.
• deep wounds type III and IV
• the wound healing is wound healing of diabetic foot ulcer, venous ulcer or pressure ulcer.
• the use is an autologous use, that is, the adipose tissue is extracted from a patient, the method of the invention is applied to said adipose tissue, and then the resulting
• decellulanzed matrix is applied to the same patient for regenerative therapy or tissue engineering purposes.
• the use is an allogenic use, that is, the adipose tissue is extracted from a first donor, the method of the invention is applied to said adipose tissue, and then the resulting
• decellularized matrix is applied to a second subject for regenerative therapy or tissue engineering purposes.
• the use is a xenogenic use, that is, the adipose tissue is extracted from a first animal donor, the method of the invention is applied to said adipose tissue, and then the resulting
• decellularized matrix is applied to a second subject which belongs to a different species as the first animal donor.
• the use in tissue engineering is for an orthopedic device.
• the matrix obtainable by the first aspect of the invention can be used as a support in any in vitro cell-culture device, thus offering a biocompatible support which fosters cell growth and differentiation.
• the second aspect of the invention can find applications for in vitro normal and diseased 2D and 3D cell culture to obtain: i) an analogous environment to in vivo tissue; ii) cell carriers and iii) organ synthesis for research, diagnostic, drug screening or implantation. It can also find
• hAT Human adipose tissue
• Tissues were cleaned with ultrapure distillated water (Millipore), fractionated manually by a blade in approximately 25 cm 3 and frozen at -30°C until use. hATs were treated for the decellularization processes as described below. All the processes were carried out under aseptic conditions.
• Tissue fractions were defrosted at room temperature (RT) and sliced manually by a blade in pieces of approximately 0.3 x 0.5 x 1 cm and treated with Trypsin and Triton-x100, Lipoprotein lipase and DNAse for tissue
• washing steps were carried out between treatments with a phosphate buffer (PBS - phosphate buffered saline) composed of Disodium hydrogen phosphate dehydrate (1 .2 mg/l,
• tissues were treated under several conditions: i) avoiding one of the treatments (trypsin and triton-x100, lipase or DNAse), ii) applying several enzyme concentration and incubation times (both lipase and DNase) and iii) applying a lower quality enzyme (DNAse for research use).
• i) avoiding one of the treatments trypsin and triton-x100, lipase or DNAse
• enzyme concentration and incubation times both lipase and DNase
• DNAse for research use A description of decellularization treatment conditions for each sample is described in Table 1 (found below).
• e 1 Description of decellularization process conditions and sample ID. Most but not all of the tissues were subjected to a pretreatment with trypsin (CTSTM TrypLETM 1 X For Use in Manufacturing Tissue-based Products, Thermofishcer Scientific) and triton-X100 (EMPROVE® bio Merck Millipore). To do so, trypsin (3 ml/tissue fraction) was added and maintained for a short period of time (30min) stirred with magnetic bars (50 rpm) at 37°C. After rising, 1 % triton-x100 in the buffer described earlier was added (8 ml/tissue fraction) and maintained overnight at RT. As indicated in Table 1 , some tissues were untreated with trypsin and tryton-x100 (M7) in order to analyze the effect of this protease and detergent treatment in the conservation of the tissue morphology and ECM proteins.
• CTSTM TrypLETM 1 X For Use in Manufacturing Tissue-based Products, Thermofishcer Scientific
• tissues were treated with a Lipoprotein lipase (IVD quality, Roche) under several enzyme concentration and incubation time (25-
• the enzyme was dissolved in the buffer described earlier (3 ml) within 0.5% triton-x100 (EMPROVE® bio, Merck Millipore). After rinsing, tissues were treated with a DNase (Benzonase EMPROVE® bio Merck Millipore) under several enzyme concentration and incubation time (714u-1428u/mg during 23h or 44h at 37°C, 100rpm. orbital shaking and vacuum). The enzyme was dissolved in the buffer described earlier (3 ml) with the addition of 2mM Magnesium chloride hexahydrate (EMPROVE® Merck Millipore) as an enzyme cofactor.
• dhATs were used freshly or lyophilized before freezing for characterization of the treatments effect on residual DNA, tissue morphology, cell-nuclei observation (Hematoxylin & Eosin) and ECM protein expression and arrangement (Immunohistochemistry). Finally some dhATs were subjected to total triglyceride and protein content quantification. Protein composition was analyzed by chromatography in one of the dAT (LM4).
• Remnant single and double strain DNA was analyzed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR).
• the DNA was extracted from the lyophilized dhATs by the QiAmp kit (Qiagen) according to the
• H&E Hematoxylin-Eosin
• dhATs were introduced in formalin (Belles Diagnostic i Investigacio), included in paraffin (Histowax LEICA) by an automatic tissue processor (ASP300, Leica Microsystems) and sectioned in 3.5 ⁇ . Some of the sections were stained by H&E following own procedures. Briefly, sections were treated with xylene (Belles Diagnostics, S.L), alcohol (Citoscan, Belles Diagnostics) and running water previous to 4-7 min Hematoxylin treatment (Haematoxylin Harris GURR® mercury free, VWR). After that, sections were treated with running water and acidic alcohol (99:1 , etanol: hidroclorhidric acid 37%, VWR) and 5-30 seg.
• Eosin (Giemsa's azur eosin methylene blue solution, VWR). Finally sections were treated with alcohol, xylene and DPX mounting media (Casa Alvarez). The immunohistochemistry was carried out for the detection of collagen type-l, collagen type-IV, laminin and heparan sulphate proteoglycan-2 (HSPG2 or perlecan) proteins by the automatic Benchmark XT (Roche Diagnostics) system. The methodology consisted on the following steps: i) deparaffination ii) antigenic recuperation, iil) dilution and incubation with appropriate
• Table 2 Specific IHQ analysis conditions for collagen type I, collagen type-IV, laminin and HSPG2.
• Triglyceride quantification was carried out by the Triglyceride Quantification Kit (Biovision) according to the manufacturer's instructions. Briefly, 100mg dhAT samples were prepared by homogenization in 1 ml solution containing 5% NP- 40 (Biovision) in water and slowly heated to 80-90°C in a water bath for 5 min. After cooling down at room temperature, the heating process was repeated one more time and insoluble material was removed by a speed centrifugation for 2min. Extracted samples were diluted 10 fold with deionized water.
• Triglyceride concentration was obtained from the standard curve and shown as relative percentage considering the non-treated original hAT as 100% triglyceride content.
• dhATs were firstly digested by cold acidic pepsin. 1 % pepsin (Sigma Aldrich) in 0.5 M Acetic acid (Panreac) solution was prepared and 1 ml/mg was added to the dhATs. The digestion was obtained under orbital shaking for 48h at RT. The acidic pH was neutralized and digested solutions were centrifuged at 4000rpm for 5min. Supernatants were stored at -30°C until use.
• Precellys®24 homogenizer (Bertin Technologies) with 1 .0 mm diameter zirconia/silica beads (BioSpec). The homogenate was sonicated for 3 min to reduce viscosity and the crude extract was then clarified by centrifugation at
• LC-MS/MS analysis was performed using a Q Exactive (Thermo Scientific) interfaced with an Easy-nLC 1000 nanoUPLC System (Thermo Scientific). Digested peptides were loaded onto an Acclaim PepMap100 precolumn (75 m x 2 cm, Thermo Scientific) connected to an Acclaim PepMap RSLC (50 ⁇ x 15 cm, Thermo Scientific) analytical column.
• Peptides were eluted directly onto the nanoES Emitter (Thermo Scientific) with a 45 min linear gradient from 3% to 30% of acetonitrile in 0.1 % of formic acid at a flow rate of 300 nl/min.
• the Q Exactive was operated in FullMS/dd-MS2 (Top10) Data Dependent Acquisition mode. Survey scans were acquired at a resolution of 70000 (m/z 200) and fragmentation spectra at 17500 (m/z 200). Peptide selection was carried out with an isolation window of 2.0 Th and normalized collision energy of 28 was applied for peptide fragmentation.
• the maximum injection time was 120 ms for survey and MS/MS scans and AGC target values of 3E6 for survey scans and 5E5 for MS/MS scans were used.
• Raw files were processed and searched with MaxQuantl (version 1 .5.3.17) software and Andromeda2 search engine.
• Precursor and fragment mass tolerances were set to 4.5 and 20 ppm respectively and up to 2 missed cleavages were allowed.
• Carbamidomethylation of Cys was set as fixed modification, oxidation of Met and protein N-term acetylation as variable modifications and a human UniProtKB-SwissProt database (version 2015_09) was used.
• a false discovery rate (FDR) 0.01 for peptides and proteins and a minimum peptide length of 7 amino acids were required. Obtained results were exported into Microsoft Office Excel (Microsoft) for further analysis.
• the mass spectrometry analysis was performed in the Proteomics Core Facility-SGIKER (member of ProteoRed-ISCIII) at the University of the Basque Country. 8 Statistical analysis
• the remnant DNA in de dhATs was significantly lower both at higher enzyme concentration (BM2 against BM4) and incubation time (BM2 against BM3 and BM4 against BM5). The differences were low (0.5-0.6 ng/mg dry weight) but up to the detection limit of the technique (20pg).
• FIG. 1 shows a comparative H&E staining of hAT and dhAT (as an example, LM4).
• hAT contains various ECM and Basement Membrane (BM) proteins, including collagen type I— VI , laminin, fibronectin, elastin, and glycosaminoglycans (GAGs).
• BM Basement Membrane
• GAGs glycosaminoglycans
• FIG. 2 shows the conservation of a very similar pattern expression of the hAT ECM and BM proteins in processed dhAT (LM4 as an example).
• Collagen type-l was expressed mainly in fibrotic region, collagen type-IV and laminin were observed mainly surrounding adipocytes (laminin was also observed in fibrillary region, not collagen type-IV).
• the specific HSPG2 protein expression pattern have also been conserved during the processing, HSPG2 expressed in the fibrillary region and less heterogeneously in the adipocyte regions.
• vascular structures have been also conserved and expressed BM proteins similarly to the original hAT.
• variations in tissue structures and protein expression were observed in dhATs obtained under different conditions of the decellularization process.
• certain lipase concentration and incubation time affected the morphology and proteins of the resultant dhATs.
• the dhAT LM5 (treated with 50u/100mg and 44h incubation at 37°C) showed alterations in tissue morphology: no visible vascular structures and destruction of ECM structure in fibrillary regions observed by H&E staining and a diminished of collagen type-l expression (FIG. 3, comparative between LM3 and LM5). No significant alterations were observed on the collagen type IV, laminin and HSPG2 expression. These results showed a limit in the lipase treatment related to the conservation of the original tissue morphology and protein expression which could be considered as the decellularization conditions lower than the LM5 dhAT.
• the effect of the trypsin/triton-x 100 pretreatment was analyzed by histologic observation of dhATs obtained with and without the pretreatment (LM3 vs. M7).
• the M7 dhAT showed a clearly higher histologic preservation of the original tissue morphology and protein expression being the most preserved dhAT analyzed in this investigation (FIG. 5).
• the trypsin treatment was very short and the triton x-100 was added in a very low percentage (1 %) these results showed a considerable adverse effect of the trypsin and non- ionic detergent treatments on the conservation of tissue morphology and ECM proteins in decellularization of hAT.
• Triglyceride quantification showed the efficacy of the lipase treatment on the delipidation of hAT observed also macroscopically (FIG. 6). Increasing concentration and incubation time of the lipase showed dhATs with
• Results of total protein content of dhATs are shown in Table 4 (found below) and showed differences between investigated dhATs. Some differences were observed in the dhATs obtained by different lipase treatments. When non-treated LM1 protein content was compared with lipase treated LM2 and LM4 dhATs a lower total protein content was observed. However in those obtained by longer lipase incubation time (LM5), a higher total protein content was observed and could be attributed to a more delipidated dhAT. More specific characterization is necessary to determine the effect of the treatments on protein content which could depend on the delipidation range.
• Table 4 Results of the total protein content of some dhAT obtained during the investigation.
• Collagens are implicated in essential biological process such as angiogenesis, basement membrane organization, blood vessel morphogenesis, cell adhesion, cell migration, collagen biosynthetic process, collagen catabolic process, endodermal cell differentiation, endothelial cell morphogenesis, epithelial cell differentiation, extracellular fibril organization, extracellular matrix disassembly, extracellular matrix organization, patterning of blood vessels, regulation of cellular component organization, skin development, wound healing, spreading of epidermial, osteoblast differentiation,
• Glycoproteins (laminin, fibulin, emilin, tenascin and fibrin protein families) are implicated in biological processes including actin cytoskeleton organization, cell adhesion, cell-matrix adhesion, collagen fibril organization, collagen metabolic process, elastic fiber assembly, endoderm development, endodermal cell differentiation, extracellular fibril organization, extracellular matrix disassembly, extracellular matrix organization, fatty acid metabolic process, regulation of cell adhesion, regulation of cell migration, regulation of embryonic development, positive regulation of epithelial cell proliferation, substrate adhesion-dependent cell spreading, epidermal growth factor receptor signaling pathway, protein localization to cell surface, regulation of cell growth, regulation of removal of superoxide radicals, secretion, triglyceride metabolic process.
• Proteoglycans are implicated in biological processes including carbohydrate metabolic process, cartilage development, chondroitin sulfate biosynthetic process, chondroitin sulfate catabolic process, chondroitin sulfate metabolic process, collagen fibril organization, dermatan sulfate metabolic process, extracellular matrix disassembly, extracellular matrix organization, glycosaminoglycan metabolic process, keratan sulfate biosynthetic process, keratan sulfate catabolic process, keratan sulfate metabolic process, organ morphogenesis, peptide cross-linking via chondroitin 4-sulfate glycosaminoglycan, positive regulation of transforming growth factor beta 1 production, response to growth factor, response to mechanical stimulus, skeletal muscle tissue development small molecule metabolic process, wound healing.
• HSPG2 Heparan sulphate proteoglycan-2
• angiogenesis brain development, carbohydrate metabolic process, cardiac muscle tissue development, cartilage development involved in endochondral bone morphogenesis, chondrocyte differentiation, chondroitin sulfate metabolic process, embryonic skeletal system morphogenesis, endochondral ossification, extracellular matrix disassembly, extracellular matrix organization, glycosaminoglycan biosynthetic process, glycosaminoglycan catabolic process, glycosaminoglycan metabolic process, lipoprotein metabolic process, phototransduction, visible light, protein localization, retinoid metabolic process, small molecule metabolic process.
• Nidogens, Periostin, Prolargin, protein s-100 are ligand, anchoring,
• phosphorylation proteins implicated in basement membrane organization, carbohydrate metabolic process, cell aging, cell adhesion, cell-matrix disassembly, establishment of protein localization to plasma membrane, extracellular matrix organization, glycosaminoglycan metabolic process, keratan sulfate biosynthetic process, keratan sulfate catabolic process, keratan sulfate metabolic process, membrane budding, membrane raft assembly, positive regulation of binding, positive regulation of cell-substrate adhesion, positive regulation of focal adhesion assembly positive regulation of GTPase activity, positive regulation of stress fiber assembly, positive regulation of substrate adhesion-dependent cell spreading, protein
• Table 5 ECM and Basal Membrane proteins conserved in dhAT (LM4) and identified by LC-MS/MS.
• composition of the obtained decellularized matrices is rich in all the components that ensure a successful exploitation in all the applications sought.
• the matrices permit their processing with several technologies: they could be successfully dissolved and freeze dryed, micronized in liquid nitrogen, compressed and rolled to obtain a porous scaffold, powder, sheet and cylindrical formats which will ideally permit to apply the material for the described applications.

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