西班牙专利ES2684856A2 Method for producing a decellularized tissue matrix

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专利摘要:
There is provided 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. There is also provided the decellularized adipose tissue obtainable by the method, further products comprising the decelluarized tissue such as coatings and scaffolds, and its uses in tissue engineering and regenerative therapy.
公开号:ES2684856A2
申请号:ES201890038
申请日:2016-12-29
公开日:2018-10-04
发明作者:Iratxe Madarieta Pardo；Nerea GARCÍA URQUIA；Rubén FERNANDEZ GARCÍA
申请人:Fundacion Tecnalia Research and Innovation；
IPC主号:

专利说明:

The present application claims the benefit of European patent application EP15203113.4 filed on December 30, 2015. The present invention relates to a method for producing a matrix of decellularized adipose tissue, to the matrix of decellularized adipose tissue obtainable by the Same and 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.
PREVIOUS TECHNIQUE
Skin ulcers are open lesions that generally involve the destruction of the epidermis and dermis. In the worst case, deep lesions also involve the destruction of the hypodermis, the lower layer of the skin. When ulcers take more than 6 weeks to heal, 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, cause a heavy burden on health systems worldwide and in many cases are not easily managed in medical offices.
Conventional topical treatments include bandages based on hydrocolloid hydrogels, foams, alginates, active carbons, silicones and silver. However, even when there is an accurate diagnosis and the usual standard treatment is administered, there is a high percentage of cases that remain resistant and do not respond positively.
One of the most promising state-of-the-art therapies for chronic skin ulcers is based on topical administration of biological matrices

acellular These matrices are products derived from animal tissue, and that can be applied topically to the wound site to stimulate and replace the altered or missing extracellular matrix (ECM). When applied topically, they provide a framework rich in proteins, glycolipids and other components of the ECM to which cells migrate and proliferate, allowing tissue regeneration. Biological matrices are currently being explored in several applications, not only in regenerative therapy but also in tissue engineering, such as internal implants and orthopedic devices. In particular, an interesting application, not related to therapy, is the use of such matrices for in vitro culture devices. These acellular matrices resemble real tissue and, therefore, can be used appropriately in any cell culture device to promote the maintenance and growth of a cell culture.
There are currently some commercial products that take advantage of these technologies in wound healing. These products are obtained from both animal (Matriderm, Primatrix, Integra) and human (Apligraf, Epicel, Graftjacket) tissues. However, they all act at the level of the epidermis and dermis. Unfortunately, matrices with the ability to regenerate hypodermis are still lacking, both because of their suboptimal properties and because they are not obtained from subcutaneous adipose tissue.
The generation of a therapeutic acellular biological matrix from an animal tissue is not a simple process for various reasons. Any method to process an initial tissue to produce an acellular matrix should ensure that the final product is endowed with a general structure and mechanical properties that resemble those of the natural extracellular matrix, it must also conserve proteins and other macromolecular components that allow that the matrix be used as a framework for regenerative processes to take place, and the most important thing is that said product must lack any nuclear component, such as nuclear waste and DNA, that makes it pro-inflammatory and immunogenic. Therefore, any method of acellular matrix production must ensure that the series of treatments applied maximizes the elimination of the immunogenic components of the original tissue and at the same time minimizes the loss of

structural characteristics that allow it to provide a barrier to infections and promote optimal cellular activity, revascularization and tissue regeneration. This balance is not easily achieved.
In the prior art various descriptions of methods for decellularizing a tissue to produce an acellular matrix are found. The majority consists of a series of consecutive treatment stages in which a variety of mechanical and / or chemical and / or biochemical (enzymatic) treatments are applied. Gilbert TW., Et. to the. “Strategies for tissue and organ decellularization” J. Cell. Biochem 2012, vol. 113, p. 2217-2222, summarize the typical treatments implemented in the decellularization of tissues in regenerative medicine. Typical enzyme treatments include trypsin, DNase, lipase and α-galactosidase. Usually, they are combined with a variety of physical and chemical treatments to facilitate the elimination of undesirable components and reinforce the effects of degrading enzymes. Chemical treatments fall into different categories, such as detergents, organic solvents, acid and alkaline solutions, etc. As disclosed in this reference, it is extremely rare to rely solely on a single chemical treatment to decellularize a tissue, and it is generally considered more advantageous to combine numerous chemical and biochemical products in a series of short washes to ensure the effectiveness of the treatments and, for therefore, an adequate decellularization. However, the use of chemicals implies a risk in terms of contamination with undesirable residues that are not suitable for biopharmaceutical applications in the final product. Ideally, it would be desirable to find methods that do not involve the use of any chemical treatment that may pose safety concerns.
Document US2012 / 0264190 discloses a decellularized and ellipidized extracellular matrix produced from adipose tissue, and methods to produce it, which can be composed of a series of treatments that include lipase slippage, digestion with proteins with a series of proteases such as pepsin, papain, matrix metalloproteases (MMP) and trypsin, digestion with nucleic acids with endonucleases, exonucleases, DNases and RNases. Without

However, this method is characterized by a stage in which detergents such as sodium deoxycholate, sodium dodecyl sulfate or Triton X-100 are used.
US2011 / 0151011 discloses a method to decellularize tissue
Adipose which comprises 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 may involve protocols in which a series of treatments can be combined, including protease treatments,
10 DNase and RNase, chelating agents such as EDTA, detergents such as Triton-X100 and lipases.
Document US2013 / 0202563 discloses a method for producing a cell growth framework from adipose tissue comprising a washing step
15 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 mainly based on the use of alcohols.
WO2011 / 132089 discloses a method for decellularizing tissues that
20 comprises contacting the original tissue with different chemical solutions and surfactants and then treating the resulting tissues treated with biochemically with a series of degrading enzymes such as nucleases.
The promising use of biological matrices for regenerative therapy guarantees the
25 development of new production methods, safer and more effective. The development of methods in which a minimum alteration of the original general structure is obtained and the maximum elimination of the proinflammatory and immunogenic components, remains an active research field.

SUMMARY OF THE INVENTION
The inventors have devised a simple but very effective method of decellularizing adipose tissue. The method of the invention comprises a reduced number of
5 stages that makes it substantially simpler than many methods found in the art, thanks to the particular conditions that have been found to be optimal in performance. The method involves the use of only two stages of biochemical (enzymatic) treatment, that is, lipoprotein lipase and a high quality nuclease (biopharmaceutical).
10 The method does not imply a series of prolonged and serial chemical treatments, which guarantees that it can be applied without raising concerns related to the safety of the final decellularized matrix. The two enzymatic treatments are carried out under conditions of concentration, duration
15 and very specific temperature, and only with enzymes of biopharmaceutical quality, which guarantees the quality of the decellularized matrix obtained. It should be noted that the method will generally involve the use of resected tissue (although it could also be applied to lipoaspirates), which does not have a combination of exhaustive chemical treatments and does not imply
20 reagents of animal origin, organic solvents or any aggressive mechanical treatment.
Therefore, a first aspect of the present invention is a method of decellularization of adipose tissue comprising the steps of: a) laminating the adipose tissue; b) treat the adipose tissue resulting from stage a) with a lipoprotein lipase at 32-42 degrees Celsius and at a concentration of 10-55 u / 100 mg, and c) treat the adipose tissue resulting from stage b) with a nuclease, at 32-42 degrees Celsius and at a concentration of 709-1433 u / mg; for a period of time necessary for the adipose tissue to have a total DNA content equal to or less than
30 50 ng / mg

In the present invention the DNA content refers to the amount of ng of DNA per mg of human fat tissue decellularized in dry weight, as described in the examples.
The decellularized matrix obtained by the method is provided with expected properties for therapeutic applications since it has a very low DNA content (usually well below 50 ng / mg, which is the threshold above which immune reactions have been described in vivo according to Crapo, PM., et. al. "An overview of tissue and whole organ decellularization processes" Biomaterials 2011, vol. 32, pp. 3233-3243), a desirable percentage of extracellular matrix protein (in particular perlecan, elastin, type I and IV collagen) and a surprisingly well-preserved general morphology. This last point is key. The matrix has a global structure that resembles that of the original tissue, which maximizes the probability of success in regenerative medicine. Therefore, the inventors have found a method that clearly establishes a balance between the removal of unwanted components, such as nuclear waste and other immunogenic DNA and the preservation of the structural integrity of the original tissue.
It should be noted that 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 may need a higher or lower content of lipids.
Therefore, a second aspect of the present invention is a matrix of decellularized adipose tissue obtainable by the method of the first aspect of the invention.
Due to the low content of undesirable components and its general morphological structure, the matrix obtainable by the method can find a myriad of different applications, including the most restrictive and demanding, such as the healing of deep wounds and ulcers that affect the hypodermis. The maximized conservation of the original morphology together with the final composition of

The treated matrix guarantees maximum performance for regenerative purposes. The matrix obtainable by the first aspect is characterized by an absence of organic solvents, low residual content in terms of immunogenic components, which guarantees a minimum response from the host when applied in therapy, structural similarities and notable functions with endogenous tissue, rich in basement membrane proteins and conserved general architecture.
A third aspect of the present invention is a powder, a foam, a particle or a hydrogel comprising the decellularized adipose tissue according to the second aspect of the invention.
A fourth aspect of the invention is a biocompatible framework or 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, the foam, the particle or the hydrogel according to the third aspect of the invention, or the biocompatible framework or coating according to the fourth aspect of the invention. invention, for use in tissue engineering and regenerative therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: sections stained with HyE of normal and decellularized TAh (TAh and TAhd-LM4). Adipocytes (column a), fibrillary regions (column b, asterisk) and vascular structures (column c, arrows) are shown. The scale bar represents 200 μm.
FIG. 2: IHC analysis of Type I collagen (fibrillar region, column a), type IV collagen (adipocyte region, column b), type IV collagen (vascular structures, column c), laminin (column d) and HSPG2 ( column e) of normal TAh and

decellularized (TAh and TAhd-LM4). Vascular structures have been identified (arrows). The scale bar represents 200 μm.
FIG. 3: staining with HyE (column a) and IHC analysis of type I collagen (column b),
5 type IV collagen (column c), laminin (column d) and HSPG2 (column e) of TAhd obtained with different lipase treatments (LM3 and LM5). The scale bar represents 100 μm or 200 μm.
FIG. 4: staining with HyE and IHC analysis of type I collagen of TAhd treated and not
10 treated with the high quality Benzasease nuclease (LM3 and M8 respectively). Column a, adipocyte region. Column b, fibrillar region. Column c, type I collagen. The scale bar represents 200 μm.
FIG. 5: staining with HyE and IHC analysis of type I (a) collagen, type IV collagen
15 (b), laminin (c) and HSPG2 (d) of TAhd not treated with trypsin / triton-x100 (M7). Adipocytic (I), fibrillary (II, asterisk) and vascular structures (III, arrow) regions are shown. For comparative purposes with treated TAhd, see Figure 3 (TAhd LM3). The scale bar represents 200 μm.
20 FIG. 6: macroscopic images and relative triglyceride content (%) of original TAh and TAhd obtained with different incubation conditions with Lipase in the decellularization process (LM1-LM5, conditions described in Table 1). The results are shown as relative triglyceride content (%) considering the original TAh as 100%.
25 FIG. 7: macroscopic images and relative triglyceride content (%) of original TAh and TAhd obtained with the decellularization process without prior treatment with trypsin / triton-x100 (M7) or treatment with Benzonasa (LM8). The results are shown as relative triglyceride content (%) considering the original TAh
30 as 100%.

FIG. 8: macroscopic and microscopic images of processed TAhd of the invention: a) macroscopic image of a powder obtained by micronization, b) microscopic image of the powder obtained by Scanning Electron Microscopy. 100 μm scale bar, c) macroscopic image of a porous framework obtained by lyophilization, d) microscopic image of a porous framework obtained by Scanning Electron Microscopy, e) macroscopic image of the plastic compression application, f) macroscopic image of a sheet obtained by plastic compression, g) macroscopic image of the rolled compressed sheet and h) macroscopic image of a cylindrical shape obtained by rolling the compressed sheet.
DETAILED DESCRIPTION OF THE INVENTION
For your understanding, the following definitions are included and are expected to be applied throughout the description, claims and drawings.
In this document the terms "acellular" and "decellularized" are used interchangeably.
The term "decellularization," as used herein, refers to a process by which a tissue undergoes one or more treatments to maximize the removal of cells present therein, leaving only the extracellular matrix (ECM) that It is rich in structural proteins such as collagen, elastin, growth factors and glycolipids.
The term "biocompatible framework", as used herein, refers to a substance with sufficient structural stability to provide a substrate to support, propitiate and promote the growth of living cells that constitute a tissue. Said framework can be used for the recovery of damaged tissue. The framework fills the space left by a wound, giving a structure to be colonized by cells and new blood vessels, which ultimately leads to tissue regeneration.

As mentioned above, a first aspect of the present invention is a method of decellularization of adipose tissue comprising the steps of: a) laminating the adipose tissue; b) treat the adipose tissue resulting from stage a) with a lipoprotein lipase at 32-42 degrees Celsius and at a concentration of 1055 u / 100 mg, and c) treat the adipose tissue resulting from stage b) with a nuclease, at 32 -42 degrees Celsius and at a concentration of 709-1433 u / mg; for a period of time necessary for the adipose tissue to have a total DNA content equal to or less than 50 ng / mg.
In a particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which treatment with lipase b) is carried out: at a concentration of 10-30 u / 100 mg for 39-49 hours at 32-42 degrees Celsius or at a concentration of 45-55 u / 100 mg for 18-28 hours at 32-42 degrees Celsius; and treatment with nuclease c) is carried out at a concentration of 709-719 u / mg for 67-77 hours at 32-42 degrees Celsius or at a concentration of 1423-1433 u / mg for 35-45 hours at 32- 42 degrees Celsius or at a concentration of 1423-1433 u / mg for 67-77 hours at 32-42 degrees Celsius.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step b) the treatment is at a concentration of 25 u / 100 mg for 44 hours at 37 degrees Celsius or at concentration of 50 u / 100 mg for 23 hours at 37 degrees Celsius, and step c) is carried out at a concentration of 709-719 u / mg for 67-77 hours at 32-42 degrees Celsius or at a concentration of 1423 -1433 u / mg for 35-45 hours at 32-42 degrees Celsius or at a concentration of 14231433 u / mg for 67-77 hours at 32-42 degrees Celsius.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step b) it is carried out at a concentration of 10-30 u / 100 mg for 39-49 hours at 32-42 degrees Celsius or at a concentration of 45-55 u / 100 mg for 18-28 hours at 32-42 degrees Celsius, and in stage c) the treatment is at a concentration of 714 u / mg

for 72 hours at 37 degrees Celsius or at a concentration of 1428 u / mg for 40 hours at 37 degrees Celsius or at a concentration of 1428 u / mg for 72 hours at 32-42 degrees Celsius.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step b) the treatment is at a concentration of 25 u / 100 mg for 4 hours at 37 degrees Celsius.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step b) the treatment is at a concentration of 50 u / 100 mg for 23 hours at 37 degrees Celsius. In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step c) the treatment is at a concentration of 714 u / mg for 72 hours at 37 degrees Celsius.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step c) the treatment is at a concentration of 1428 u / mg for 40 hours at 37 degrees Celsius.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step c) the treatment is at a concentration of 1428 u / mg for 72 hours at 37 degrees Celsius.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue further comprises a stage between stages a) and b), in which the adipose tissue resulting from step a) is treated with trypsin and triton-X100 . In another particular embodiment, the last stage is characterized by

that the trypsin-tritonX100 treatment further comprises EDTA and is carried out at 37 degrees Celsius overnight and the Triton-X100 is 1% in v / v (1 ml / 100 ml).
This additional stage is not mandatory, as can be seen in the experimental data indicated below, where the decellularized matrix with the best properties is obtained without it (M7). However, if you want the final product to have a lower lipid content (such as that found for LM3 or LM4), then this optional treatment step of trypsin + triton-X100 can be used.
In another particular embodiment of the first aspect of the invention, the method further comprises a step d) comprising freezing or lyophilizing and sterilizing the adipose tissue resulting from step c).
In another particular embodiment of the first aspect of the invention, sterilization is carried out with ethylene dichloride. In another particular embodiment, sterilization is carried out with ultraviolet light. In another particular embodiment, the method is carried out under aseptic conditions.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in stages b) and c) the treatments are vacuum treatments and with stirring at 100-150 rpm.
In another particular embodiment, agitation at 100-150 rpm is orbital agitation.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which after steps a), b) and c), there is a buffer washing step at room temperature, under vacuum and with stirring at 100-150 rpm, and in which the washing stage after stage a), the buffer additionally comprises at least one antibiotic, at least one antifungal and at least one protease inhibitor, in the washing stage after from

step b) the buffer comprises at least one antibiotic, at least one antifungal, 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 an antibiotic, at least one antifungal, at least one protease inhibitor and at least one nuclease inhibitor.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step b), the treatment with lipoprotein lipase comprises 0.5% Triton-X100 and a cofactor in buffer of phosphate. In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step b), the treatment with lipoprotein lipase comprising 0.1% Triton-X100 and a buffer cofactor phosphate.
In another particular embodiment, steps b) and c) are carried out in the presence of a cofactor. In another particular embodiment, the cofactor is magnesium (Mg + 2).
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step a), the lamination of adipose tissue is manual. In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step a), the laminated block has a size of 1-15 cm in length, 0.5-8 cm wide and 0.3-2 cm thick.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step b), lipoprotein lipase is selected from the group consisting of lipase and phospholipase of bacterial origin (Pseudomonas genera, Staphyloccus, Bacillus, etc.), of yeast origin (species Candida aldicans, Candida Antarctica, Candida rugosa, Geotrichum asteroids, Geotrichum candidium, Trichosporon fermentans, Saccharomycopsis lipolytica, Yarrowia lipolytica), of fungal origin (genera Penicillu, Rhomopuscorus, Rhomopuscorus, Rhomopus, Rhomopus, Rhomopus, Rhomoporiz etc.) and of mammalian origin (pig, bovine,

equine, human, etc.). The latter group includes lipoprotein lipase, acylglycerol lipase, triacylglycerol lipase, a hormone-sensitive lipase, pancreatic lipase, bile salt activated lipase, protein 1 related to pancreatic lipase, protein 2 related to pancreatic lipase, phospholipase A2, calcium independent phospholipase A2, endothelial lipase, phosphatidylinositol phospholipase A2, endogenous phospholipase C, phosphoinositide phospholipase C, phospholipase C, lysophospholipase D and phospholipase D.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step c), the nuclease is Benzonasa®. Benzonasa is an endonuclease with very high pharmaceutical quality and is supplied by Merck-Millipore, which in its technical specifications indicates: Benzonasa® is a unique endonuclease, modified by genetic engineering, which is only available in Merck Millipore. Produced in E. coli, this nonspecific recombinant endonuclease cleaves all types of DNA and RNA variants into fragments that comprise <8 soluble base pairs. This leads to a minimum load of nucleic acid.
In another particular embodiment of the first aspect of the invention, the method of decellularization of adipose tissue is a method in which in step c), the nuclease is selected from the group consisting of nucleases of bacterial origin (Serratia marcescens, Clostridium), of yeast origin, fungal origin (Asperguillus) and mammalian origin (pig, bovine, equine, human, etc.) including deoxyribonucleases (DNases) and ribonucleases (RNases) such as DNase I, DNase II, DNase V, micrococcal nuclease Type-specific site DNase, RNase I, RNase H, RNase III, RNase L, RNase P, nuclease S1, RNase yeast, RNase U2, RNase T2, RNase T1, RNase P4, RNase M5, RNase IX, RNase E, RNase D, RNase alpha, RNase PH, RNase AS, RNase Fitb, RNase J1, RNase pancreatic, nanoRNase.
As mentioned above, a second aspect of the present invention is a matrix of decellularized adipose tissue obtainable by the

method of the first aspect of the invention.
In a particular embodiment of the second aspect of the invention, the adipose tissue to be treated is of subcutaneous origin, such as breast or abdominal.
In a particular embodiment of the second aspect of the invention, the adipose tissue to be treated is obtained from lipoaspirate.
In another particular embodiment of the second aspect of the invention, the adipose tissue to be treated is obtained by resection.
In another particular embodiment of the second aspect of the invention, the adipose tissue comes from the omentum or omentum.
In a particular embodiment of the second aspect of the invention, the adipose tissue is mammalian adipose tissue.
In a particular embodiment of the second aspect of the invention, the adipose tissue is human adipose tissue.
In a particular embodiment of the second aspect of the invention, 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 since it is well below the 50 ng / mg threshold described above. Therefore, the first aspect of the invention allows a decellularized matrix to be obtained which, although characterized by a highly conserved morphology compared to the original tissue, has a nuclear residual content that minimizes the likelihood of adverse immune reactions.
in vivo
In a particular embodiment of the second aspect of the invention, the adipose tissue matrix further comprises exogenous bioactive molecules. In a

Particular embodiment of the second aspect of the invention, the natural or synthetic bioactive molecules are selected from the group consisting of growth factors, hormones, vitamins, antioxidant agents, anti-inflammatory, antibacterial, antifungal, accelerators / wound healing promoters, or mixtures of the same.
In a particular embodiment of the second aspect of the invention, the adipose tissue matrix further comprises i) synthetic polymers (PEG, poly (α-hydroxy esters), polystyrene, polyurethane and copolymers and mixtures) and ii) natural polymers (protein based , elastin, collagen, fibrin; or based on polysaccharides, collagen, chitosan, hyaluronic acid, beta-glucans, gelatin, microcellulose and nanocellulose and their derivatives).
In a particular embodiment of the second aspect of the invention, the matrix of adipose tissue additionally comprises living cells of human or animal (within or above the material): somatic cells (adipocytes, fibroblasts, osteocytes, osteoblasts, chondrocytes, chondroblasts, myoepithelium , bone marrow, macrophages, etc.), endothelial cells, stem cells or induced pluripotent stem cells.
In a particular embodiment of the second aspect of the invention, the decellularized adipose tissue has a total protein content of 100-931 μg / mg and a triglyceride content of 25-65% of the original tissue (w / w).
Depending on the final application, the second aspect of the invention can be further processed by micronization, plastic compression, dissolution, freeze-drying, self-assembly, chemical cross-linking and / or induced physical interactions, electro-spinning, rotating coating or 3D printing.
As mentioned above, a third aspect of the invention is a powder, a foam, a particle or a hydrogel comprising the decellularized adipose tissue according to the second aspect of the invention.

In a particular embodiment of the third aspect of the invention, the powder, foam, particle or hydrogel comprising the decellularized adipose tissue is mixed with other bioactive components such as growth factors, exogenous structural proteins or healing agents. The resulting composite mixture has additional advantages in terms of tissue regeneration.
As mentioned above, a fifth aspect of the invention is the decellularized adipose tissue according to the second aspect of the invention, the powder, the foam, the particle or the hydrogel according to the third aspect of the invention, or the biocompatible framework or the coating. biocompatible according to the fourth aspect of the invention for use in tissue engineering and regenerative therapy.
In a particular embodiment of the fifth aspect, the use is in combination with a matrix selected from the group consisting of Matriderm, Primatrix, Integra, Apligraf, Epicel and Graftjacket.
In a particular embodiment of the fifth aspect, the use is in combination with any other matrix available in regenerative medicine.
In a particular embodiment of the fifth aspect, the therapy is a wound healing therapy. In particular, the multiple applications of the first aspect of the invention include: wound healing, tissue engineering, regenerative medicine, additive therapies and cellular supports for the treatment of diseases or damaged tissues and organs, healing or disease prevention and restoration, correction or alteration of the physiological functions of: i) connective tissue (adipose tissue, skin, blood vessels, cartilage, bone, etc.), ii) epithelial and endothelial tissue (epidermis, intestinal epithelium, vascular endothelium, etc.), iii) muscle tissue and iv) nerve tissue.
In a particular embodiment, wound healing is wound healing of deep wounds (type III and IV) of the hypodermis. In a particular embodiment, wound healing is the healing of foot ulcer wounds

diabetic, venous ulcer or pressure ulcer.
In a particular embodiment of the fifth aspect, the use is an autologous use, that is, adipose tissue is removed from a patient, the method of the invention is applied to said adipose tissue and then the resulting decellularized matrix is applied to the same patient for purposes. of regenerative therapy or tissue engineering.
In a particular embodiment of the fifth aspect, the use is an allogeneic use, that is, adipose tissue is removed 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 the purpose of regenerative therapy or tissue engineering.
In a particular embodiment of the fifth aspect, the use is a xenogenic use, that is, adipose tissue is removed 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 that belongs to a different species from that of the first animal donor.
In a particular embodiment of the fifth aspect, the use in tissue engineering is for an orthopedic device.
The use of the second aspect of the invention for in vitro cell culture is also part of the invention. The matrix that can be obtained 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 that promotes cell growth and differentiation.
Therefore, the second aspect of the invention can find applications for normal and diseased 2D and 3D cell culture in vitro to obtain: i) an environment analogous to tissue in vivo; ii) cell transporters and iii) organ synthesis for research, diagnosis, drug detection or implantation. You can also find applications in bandage materials, conveyors

Cellular, coatings, fillers, fluid powder particles for the administration of drugs, biotintas, frames (foams, micro and macro porous hydrogels, thermoinducting hydrogels), sheets, cylindrical or tubular patches, spraying.
Throughout the description and claims, the word "comprises" and variations of the word are not intended to exclude other technical characteristics, additives, components or steps. In addition, the word "comprises" and its variations include the expression "consisting of". Additional objects, advantages and features of the invention will be obvious to those skilled in the art after examining the description or can be learned by practicing the invention. The following examples are provided as illustrative and are not intended to limit the present invention. In addition, the present invention includes all possible combinations of particular and preferred embodiments described herein.
EXAMPLES
Materials and methods
1 Donation of human adipose tissue
Human adipose tissue (TAh) was donated by patients who underwent plastic surgery at the Polyclinic hospital of Guipúzcoa according to law 1301/2006 of the Ministry of Spain of November 10, which establishes quality and safety standards for donation, acquisition , assay, processing, preservation, storage and distribution of human cells and tissues. The research was carried out with the prior approval of the Ethical Commission of the Basque Country (Clinical Research Ethics Committee, CEIC).
The tissues were cleaned with ultrapure distilled water (Millipore), fractionated manually with a blade at approximately 25 cm3 and frozen at 30 ° C until use. TAhs were treated following the decellularization processes

described below. All processes were performed under aseptic conditions.
2 Decellularization of human adipose tissue Tissue fractions were thawed at room temperature (RT) and
5 manually cut with a knife into pieces of approximately 0.3 x 0.5 x 1 cm and treated with Trypsin and Triton-x100, Lipoprotein lipase and DNase for tissue decellularization.
To eliminate residual reagents, among the treatments, they were carried out
10 washing steps with a phosphate buffer (PBS - phosphate buffered saline) composed of hydrogen disodium phosphate dihydrate (1.2 mg / l, EMPROVE® bio, Merck Millipore), sodium dihydrogen phosphate dihydrate (0.885 mg / l, EMPROVE® bio, Merck Millipore), penicillin-streptomycin 10 ml / l (according to cGMP, LONZA), fungizone 10 ml / l (Fungizone® Antifungal, ThermoFisher Scientific) and cocktail
15 protease III 1.25 ml / l inhibitor (without EDTA, Merck Millipore). For washing, to each tissue fraction, 5 ml of fresh phosphate buffer was added and kept under vacuum and with orbital stirring (100 rpm) at RT for 10 min. This rinsing step was repeated at least 5 times and could include cofactors for enzyme inactivation.
20 To adjust the decellularization process and analyze the effect of each treatment on the effectiveness of the decellularization and conservation of native MEC proteins, the tissues were treated under various conditions: i) avoiding one of the treatments (trypsin and triton- x100, lipase or DNAse), ii) applying various
25 enzymatic concentrations as well as incubation times (both lipase and DNAse) and iii) applying a lower quality enzyme (DNAse for research use). A description of the decellularization treatment conditions for each sample is shown below in Table 1.

DECELULARIZATION CONDITIONS
ID TRIPSINE + TRITON-X100LIPASSDNASA
BM1 30 min. Trypsin / EDTA at 37 ° C and 1% Triton-x overnight at RT50 u./100 mg 23 h 37 ºCWITHOUT TREATING
BM2 Benzonasa EMPROVE® bio 714 u./mg 40 h. / 37 ºC
BM3 EMPROVE® bio benzene 714 u./mg 72 h. / 37 ºC
BM4 EMPROVE® bio benzene 1428 u./mg 40 h. / 37 ºC
BM5 EMPROVE® bio benzene 1428 u./mg 72 h. / 37 ºC
BM6 50 u / 100 mg 23 h 37 ºCDNAse for research use 1428 u./mg 72 h. / 37 ºC
LM1 WITHOUT TREATINGEMPROVE® Bio Benzonease (Merck Millipore) 1428 u./mg 72 h. / 37 ºC
LM2 25 u / 100 mg 23 h 37 ºC
LM3 25 u / 100 mg 44 h 37 ºC
LM4 50 u / 100 mg 23 h 37 ºC
LM5 50 u / 100 mg 44 h 37 ºC
M7 WITHOUT TREATING25 u / 100 mg 44 h 37 ºCEMPROVE® bio benzene 1428 u./mg 72 h./37 ºC
M8 30 min. T / E at 37 ° C and 1% Triton-x overnight at RTWITHOUT TREATING

Table 1: Description of the decellularization process conditions and sample ID
Most tissues, although not all, underwent prior trypsin treatment (CTS ™ TrypLE ™ 1X for use in the manufacture of tissue-based products, Thermofishcer Scientific) and Triton-X100 (EMPROVE® bio Merck Millipore) . For this, trypsin (3 ml / tissue fraction) was added and kept for a short period of time (30 min) stirred with magnetic bars (50 rpm) at 37 ° C. After rinsing, 1% triton-x100 was added in the buffer described above (8 ml / tissue fraction) and kept overnight at RT. As indicated in Table 1, some tissues were not treated with trypsin or triton-x100 (M7) in order to analyze the effect of this protease and detergent treatment in the preservation of tissue morphology and proteins. the MEC.
After rinsing, the tissues were treated with a Lipoprotein lipase (IVD quality, Roche) at various enzyme concentrations and incubation times (2550 u / 100 mg for 23 h or 44 h at 37 ° C, 100 rpm, with orbital agitation and vacuum, Table 1). The enzyme was dissolved in the buffer described above (3 ml) in 0.5% tritonx100 (EMPROVE® bio, Merck Millipore).
After rinsing, the tissues were treated with a DNase (EMPROVE® bio Merck Millipore benzene) at various enzyme concentrations and incubation times (714 u-1428 u / mg, for 23 or 44 hours, at a temperature of 37 ° C and 100 rpm, with orbital and vacuum agitation). The enzyme was dissolved in the buffer described above (3 ml) with the addition of 2 mM magnesium chloride hexahydrate EMPROVE® Merck Millipore) used as an enzymatic cofactor. Some tissues were treated with quality DNAse I for research use (Sigma Aldrich Chemical) under specific conditions of Lipase and DNAse (50 u / 100 mg and 714 u / mg at 72 h respectively).
Recent TAhd were used or freeze-dried before freezing to characterize the effect of treatment on residual DNA, tissue morphology,

observation of cell nuclei (Hematoxylin and Eosin) and protein expression and disposition of the MEC (immunohistochemistry). Finally, some TAhd underwent quantification of the total triglyceride and protein content. The protein composition was analyzed by chromatography in one of the TAd (LM4).
3 Residual DNA quantification Residual single and double stranded DNA was analyzed by Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR). The DNA was extracted from freeze-dried TAhd with the QiAmp kit (Qiagen) according to the manufacturer's instructions. For absolute quantification, a standard curve of human blood DNA samples of known concentration was obtained. To do this, DNA extraction was performed with the Qiamp DNA Blood MiniKit (Qiagen) according to the manufacturer's instructions. A DNA solution of 50 ng / ml was obtained and analyzed by an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA) and serial dilutions were prepared. Detection was performed through the specific sequence of the human beta hemoglobin gene (HBB, 11p.15.5) and analyzed using LightCycler 480II (Roche).
The reactions were performed in triplicate; the data were shown as residual DNA (ng) by TAhd in dry weight (mg) and subjected to statistical analysis as described below. The detection limit of the technique was calculated in approximately 20 pg of DNA. DNA quantification was performed in DNAdata, a Diagnostic Center for Genetic Diseases authorized by the Government of the Basque Country.
4 Histological analysis The recent, preserved TAhd were subjected to histological analysis of tissue morphology and observation of cell nuclei by staining with Hematoxylin and Eosin (HyE) and immunohistochemical analysis of MEC proteins.
For this, TAhd were introduced in formalin (Bellés Diagnóstic i Investigació), were included in paraffin (Histowax LEICA) with an automatic tissue processor

(ASP300, Leica Microsystems) and were sectioned to a size of 3.5 μm. Some of the sections were stained with HyE following own procedures. In summary, the sections were treated with xylene (Bellés Diagnostics, S.L), alcohol (Citoscan, Bellés Diagnostics) and running water before hematoxylin treatment for 4
5 7 min. (Harris GURR® Hematoxylin without mercury, VWR). After this, the sections were treated with running water and acid alcohol (99: 1, ethanol: 37% hydrochloric acid, VWR) and Eosin for 5-30 seconds (Giemsa methylene blue azos eosin solution, VWR). Finally, the sections were treated with alcohol, xylene and DPX mounting media (Casa Álvarez).
10 Immunohistochemistry was performed for the detection of type I collagen, type IV collagen, laminin and heparan sulfate 2 proteoglycan (HSPG2 or perlecan) proteins with the automatic Benchmark XT (Roche Diagnostics) system. The methodology consisted of the following stages: i) dewaxed ii) recovery
Antigenic, iii) dilution and incubation with an appropriate monoclonal antibody, iv) amplification, detection and visualization with the Amplification Kit (Roche) and the Ultraview Universal DAB Detection Kit (Roche). Staining was optimized for each protein using as controls kidney, skin, colon, placenta and adipose tissue and the specific conditions detailed in Table 1. Samples were
20 visualized with a BX-51 microscope (Olympus) and digitized using an RS Nanozoomer 2.0 RS (Hamamatzu) scanner. Table 2 (below) describes the specific IHC conditions for each protein.
IHC conditions and controls
Protein Antigenic recoveryAntibody, dilution and incubationPositive control
Type I collagen Cellular conditioning solution 1 (CC1, Roche) ph = 8.3 30 min.AntibodyMouse monoclonal antibody against collagen type I clone COL-I (Abcam)Kidney
Dilution 1: 200
Incubation 37 ° C / 32 min

IHC conditions and controls
Protein Antigenic recoveryAntibody, dilution and incubationPositive control
Type IV collagen CC1 (pH = 8.3) 30 min.AntibodyMouse monoclonal antibody against collagen type IV clone CIV22 (Roche)Kidney
Dilution No dilution is required.
Incubation 37 ° C / 32 min
Laminin Pepsin-0.2 M HCl (Sigma) TA 30 min.AntibodyMouse monoclonal antibody against human laminin, clone 4C7 (Dako)Kidney
Dilution 1:10
Incubation TA / 60 min
HSPG2 / Perlecan CC1 (pH = 8.3) 30 min.AntibodyMouse monoclonal antibody against heparan sulfate proteoglycan 2, clone A74, (Abcam)Colon
Dilution 1:10
Incubation 37 ° C / 32 min
Table 2: Specific IHC analysis conditions for type I collagen, type IV collagen, laminin and HSPG2.
5 These histological evaluations were performed at Althia Health, S.L. (Althia), a company accredited by the National Accreditation Entity (ENAC) of Spain for pathology and Molecular Pathology tests and as a technical competence according to the criteria of the UNE-EN-ISO 15189: 2007 standard (reference number of
10 laboratory 1029 / LE2012).
5 Quantification of triglyceride content Quantification of triglycerides was performed with the Quantification Kit

Triglycerides (Biovision) according to the manufacturer's instructions. In summary, 100 mg TAhd samples were prepared by homogenization in a 1 ml solution containing 5% NP-40 (Biovision) in water and slowly heated at 80-90 ° C in a water bath for 5 min. After cooling to room temperature, the heating process was repeated once more and the insoluble material was removed by centrifugation with speed for 2 min. The extracted samples were diluted 10 times with deionized water. Absorbance (570 nm) was measured in triplicate in samples and triglyceride standards in a multi-well spectrophotometer (Power Wave Xs, Biotek). The triglyceride concentration was obtained from the standard curve and is shown as a relative percentage considering that the original untreated TAh is 100% triglyceride content.
6 Quantification of total protein content For quantification of total protein, TAhd were first digested with cold acid pepsin. 1% pepsin (Sigma Aldrich) in 0.5 M acetic acid solution (Panreac) was prepared and 1 ml / mg was added to the TAhd. Digestion was obtained with orbital agitation for 48 h at RT. The acidic pH was neutralized and the digested solutions were centrifuged at 4000 rpm for 5 min. Supernatants were stored at -30 ° C until use.
Total protein content was analyzed with the PierceTM BCA Protein Assay Kit (Thermoficher Scientific) according to the manufacturer's instructions. A standard curve was obtained with bovine serum albumin (BSA) patterns included in the kit. The absorbance (580 nm) was measured in triplicate in samples and standards in a multi-well spectrophotometer (Power Wave Xs, Biotek). The results are shown as protein content (μg) per dry TAhd (mg).
7 Liquid chromatography with tandem mass spectrometry analysis (LC-MS / MS) The protein composition of a TAhd (LM4) was analyzed by LC-MS / MS. For protein extraction, TAhd was homogenized in 8 M urea using a

Precellys®24 homogenizer (Bertin Technologies) with 1.0 mm diameter zirconia / silica beads (BioSpect). The homogenate was subjected to ultrasound for 3 min to reduce viscosity and the crude extract was then clarified by centrifugation at 16,000 x g for 10 min, transferred to new tubes and stored at -20 ° C. After this, protein digestion was performed by diluting the sample 5 times with 50 mM NH4HCO3. In summary, the proteins were reduced (5 mM DTT, room temperature, 25 min), alkylated (15 mM iodoacetamide, room temperature, 30 min) and digested with trypsin (0.01 μg / μl, 37 ° C, 16 hours , Roche Diagnostics). The resulting peptides were desalinated using Micro Spin C-18 Columns (Harvard Team).
LC-MS / MS analysis was performed using a Q Exactive (Thermo Scientific) interconnected with an Easy-nLC 1000 nanoUPLC System (Thermo Scientific). The digested peptides were loaded into an Acclaim PepMap100 pre-column (75 μm x 2 cm, Thermo Scientific) connected to an Acclaim PepMap RSLC analytical column (50 μm x 15 cm, Thermo Scientific). The peptides were eluted directly on the nanoES emitter (Thermo Scientific) with a linear gradient of 3% to 30% acetonitrile for 45 min in 0.1% formic acid at a flow rate of 300 nl / min. The Q Exactive worked in a FullMS / dd-MS2 Data Dependent Acquisition mode (Top10). The study scanners were acquired at a resolution of 70,000 (m / z 200) and the fragmentation spectra at 17500 (m / z 200). Peptide selection was performed with a 2.0 Th isolation window and a standardized collision energy of 28 was applied for peptide fragmentation. The maximum injection time was 120 ms for study and MS / MS scanners and target AGC values of 3E6 were used for the study scanners and 5E5 for the MS / MS scanners. The original files were processed and investigated with the MaxQuant1 software (version 1.5.3.17) and the Andromeda2 search engine. Mass precursor and fragment tolerances were set at 4.5 and 20 ppm respectively and up to 2 skips omitted were allowed. Cys carbamidomethylation was established as fixed modification, Met oxidation and acetylation in the N terminal of the protein as variable modifications and a human UniProtKB-SwissProt database (version 2015_09) was used. Be

required a false discovery rate (FDR) of 0.01 for peptides and proteins and a minimum peptide length of 7 amino acids. The results obtained were exported to Microsoft Office Excel (Microsoft) for further analysis.
5 The mass spectrometry analysis was performed at the Proteomics Core Facility-SGIKER (member of ProteoRed-ISCIII) at the University of the Basque Country.
8 Statistical analysis
10 The quantitative results were referred to as mean típica standard deviation and a statistical analysis of the residual DNA data was performed using the Student t test (p> 0.05, p ≥ 0.01 and p ≥ 0.001).
Results and Comments
15 1 Quantification of residual DNA
All TAhd analyzed, except BM1 (not treated with Benzonasa), showed a very low residual DNA (Table 3, below) and met the criteria
20 general decellularization (DNA ≤ 50 ng / mg), a threshold above which immune reactions have been described in vivo (Crapo PM., Et. Al., Mentioned above). BM1 showed a dry weight of 365,506 ± 78,105 ng / mg while the TAhd obtained with different conditions of incubation of Benzonasa, showed a residual DNA between 0.004-0.613 ng / mg in dry weight (BM2-BM5). He
25 residual DNA in the TAhd was significantly lower both at higher enzymatic concentration (B2 vs. BM4) and at longer incubation time (BM2 vs. BM3 and BM4 vs. BM5). The differences were low (0.5-0.6 ng / mg dry weight) although up to the limit of detection of the technique (20 pg).
RESIDUAL DNA
ID AVERAGE  TYPICAL DEVIATION ng of DNA / mg of TAhd in dry weightStudent's T vs. BM1Student's T vs. BM2
BM1 356,506 ± 78,102*

RESIDUAL DNA
ID AVERAGE  TYPICAL DEVIATION ng of DNA / mg of TAhd in dry weightStudent's T vs. BM1Student's T vs. BM2
BM2 0.613 ± 0.050*
BM3 0.004 ± 0.002***
BM4 0.109 ± 0.015***
BM5 0.047 ± 0.005***
BM6 4,150 ± 0.187Student's T vs. BM3
**
LM1 0.0002 ± 0.0003Student's T vs. LM1
LM3 0.306 ± 0.005**
LM4 0.045 ± 0.017*
LM5 0.212 ± 0.023**
M7 0.015 ± 0.004*
Table 3: Results of the quantification of residual DNA in TAhd. The statistical analysis of Student's t of the significant differences between the residual DNA obtained with different decellularization conditions * (p> = 0.01) and ** (p> = 0.001).
5 When the qualities of the reagents were modified, greater differences were found in the residual DNA. The treatments with quality DNAse (BM6) for research and Benzonasa Emprove BIO (BM3) were compared and BM6 showed a statistically significant lower residual DNA of 4,146 ng / mg dry weight
10 (Table 3).
Residual DNA in TAhd also showed differences according to lipase treatment. LM1 (not treated with Lipase) showed a statistically significant lower residual DNA (0.2-0.3 ng / mg) compared to LM3 and LM5 (treated for 44 h
15 with 25 u / 100 mg and 50 u / 100 mg respectively). This effect could be attributed to interactions between enzymes in decellularization processes. When a 23 h treatment with lipase (LM4) was applied, the effect of the enzyme on the residual DNA was irrelevant (0.045 ng / mg).
20 TAhd not treated with trypsin or triton-x100 (M7) showed a statistically lower residual DNA (0.015 ng / mg) than that obtained with previous treatment with trypsin and triton-x100 (LM3, 0.306 ng / mg). The differences were low (0.29 ng / mg)

although up to the limit of detection of the technique (20 pg).
2 Histological analysis and IHC Morphologically, staining with HyE of the TAhd showed a mature adipose tissue in which the absence of cell nuclei stands out, but where the conservation of the tissue structure is observed: the cytoplasmic space of the adipocytes is recognized and the denser fibrous tissue as well as vascular structures. FIG. 1 shows a comparative staining with HyE of TAh and TAhd (as an example, LM4). TAh contains several proteins of the MEC and Basal Membrane (MB), including collagen type I-IV, laminin, fibronectin, elastin and glycosaminoglycans (GAG). In this investigation, type I collagen, type IV collagen, laminin and a MB specific heparan sulfate proteoglycan (HSPG2) have been analyzed by IHC. FIG. 2 shows the conservation of a very similar expression pattern of the proteins of the MEC and the MB of TAh in processed TAhd (LM4 as an example). Type I collagen was expressed primarily in the fibrotic region, type IV collagen and laminin were mainly observed around adipocytes (laminin was also observed in the fibrillar region, not type IV collagen). The specific expression pattern of the HSPG2 protein has also been preserved during processing, HSPG2 was expressed in the fibrillar region and less heterogeneously in the adipocytic regions. Finally, vascular structures have also been conserved and MB proteins were similarly expressed in the original TAh.
However, variations in tissue structures and protein expression in TAhd obtained under different conditions of the decellularization process were observed. In relation to the lipase treatment, a certain lipase concentration and the incubation time affected the morphology and the resulting TAhd proteins. In particular, TAhd LM5 (treated with 50 u / 100 mg and 44 h of incubation at 37 ° C) showed alterations in tissue morphology: non-visible vascular structures and destruction of the structure of the ECM in the fibrillary regions observed by staining with HyE and decreased expression of type I collagen (FIG. 3, comparative between LM3 and LM5). I dont know

they observed significant alterations in the expression of type IV collagen, laminin and HSPG2. These results showed a limit in lipase treatment related to the conservation of the original tissue morphology and protein expression, which could be considered as the decellularization conditions were lower than those of TAhd LM5.
There were also some differences related to the treatments with nuclease (Benzonasa). In TAhd M8 (not treated with Benzonasa) some cell nuclei were observed in both adipocytic and fibrous regions. As shown with lipase treatment, Benzonase also produces alterations in the fibrillar structures of the tissue and a decrease in the expression of type I collagen (FIG. 4). No significant alterations were observed in the expression of type IV collagen, laminin and HSPG2. These results showed that the treatment with Benzonasa should be determined by the residual DNA (in our investigations, the limit shown in the treatment with Benzonasa was the conditions of TAhd BM3).
The effect of previous treatment with trypsin / triton-x 100 was analyzed through histological observation of TAhd obtained with and without prior treatment (LM3 vs. M7). TAhd M7 showed a clearly superior histological conservation of the original tissue morphology and protein expression, with TAhd analyzed in this investigation being the most conserved (FIG. 5). Although trypsin treatment was very short and triton x-100 was added in a very low percentage (1%), these results showed a considerable adverse effect of treatments with non-ionic detergents and trypsin on the conservation of tissue morphology and MEC proteins in the decellularization of TAh.
3 Quantification of the triglyceride content The quantification of triglycerides showed the efficacy of lipase treatment on the TAh gliding also observed macroscopically (FIG. 6). The increase in the concentration and incubation time of lipase showed a TAhd with a decreased triglyceride content (ranging from 70 10% to 15  10%) in

comparison with the original TAh (considered as 100% triglyceride content). The results also showed differences between untreated TAhd with a previous trypsin / triton-x 100 treatment and treatment with Benzonasa. The absence of these treatments results in a lower amount of TAhd
5 slipped (LM3 vs. M7).
4 Quantification of total protein content Table 4 (below) shows the results of the total protein content of TAhd and shows the differences between the investigated TAhd. Be
10 observed some differences in the TAhd obtained by different treatments with lipase. When the untreated LM1 protein content was compared with Lh2 and LM4 TAhd treated with lipase, a lower total protein content was observed. However, in those obtained during a longer incubation time with lipase (LM5), a higher total protein content was observed and
15 could be attributed to a more tarnished TAhd. A more specific characterization is needed to determine the effect of the treatments on the protein content that would depend on the gliding interval.
TOTAL PROTEIN CONTENT
ID AVERAGE  TYPICAL DEVIATION (μg of protein / mg of TAhd in dry weight)
LM1 759.1 ± 17.0
LM2 565.2 ± 22.3
LM3 931.5 ± 21.5
LM4 693.6 ± 6.5
LM5 820.6 ± 14.5
M7 796.3 ± 20.6
M8 824.12 ± 12.5
20 Table 4. Results of the total protein content of some TAhd obtained during the investigation

5.-LC-MS / MS Analysis Specific protein analysis was carried out for TAhd LM4. The results showed a composition of 136 human proteins conserved after the decellularization process (false discovery rate: 1% and discarding the proteins detected by 1 peptide).
Table 5 (below) shows some of the proteins conserved after the decellularization process with special relevance in the composition, interactions and assembly of MEC and MB proteins. The results include collagen (I, IV, VI, XV and XVIII), glycoproteins (families of the proteins laminin, fibulin, emilin, tenascin and fibrin), proteoglycans (lumicano, decorin, mimecano and biglican) and a specific sulfated proteoglycan of the MB (HSPG2). Other ligands have also been identified as well as phosphorylation and anchor proteins (Nidogen, Periostin, Prolargine, s-100 protein).
Collagens are involved in essential biological processes such as angiogenesis, basal membrane organization, blood vessel morphogenesis, cell adhesion and migration, collagen biosynthetic process, collagen catabolic process, endodermal cell differentiation, endothelial cell morphogenesis, cell differentiation epithelial, organization of extracellular fibrils, disassembly of the extracellular matrix, organization of the extracellular matrix, modeling of blood vessels, regulation of the organization of cellular components, skin development, wound healing, epidermal dissemination, osteoblast differentiation, homeostasis of various cells within a tissue, adhesion between cells of a single organism, organ morphogenesis, positive regulation of cell proliferation, response to hydrostatic pressure.
Glycoproteins (families of the proteins laminin, fibulin, emilin, tenascin and fibrin) are involved in biological processes, including the organization of the actin cytoskeleton, cell adhesion, adhesion of the cell matrix, organization of collagen fibrils, process metabolic of collagen,

elastic fiber assembly, endoderm development, cell differentiation of the endoderm, organization of extracellular fibrils, disassembly of the extracellular matrix, organization of the extracellular matrix, metabolic process of fatty acids, regulation of cell adhesion, regulation of cell migration, embryonic development regulation, positive regulation of epithelial cell proliferation, cell dissemination dependent on substrate adhesion, epidermal growth factor receptor signaling pathway, cell surface protein localization, cell growth regulation, regulation of elimination of superoxide radicals, secretion, metabolic process of triglycerides.
Proteoglycans (lumicano, decorina, mimecano and biglican) are involved in biological processes, including metabolic processes of carbohydrates, cartilage development, biosynthetic process of chondroitin sulfate, catabolic process of chondroitin sulfate, metabolic process of chondroitin sulfate, organization of collagen fibrils, metabolic process of dermatan sulfate, disassembly of the extracellular matrix, organization of the extracellular matrix, metabolic process of glucosaminoglycans, biosynthetic process of keratan sulfate, catabolic process of keratan sulfate, metabolic process of keratan sulfate, organ morphogenesis, cross-linking of peptides through glucosaminoglycan chondroitin 4-sulfate, positive regulation of the production of transforming growth factor beta 1, response to growth factors, responses to mechanical stimuli, in the development of musculoskeletal tissue, in the process m etabolic of small molecules and wound healing.
The heparan sulfate 2 proteoglucan (HSPG2) specific to BM, is involved in angiogenesis, in the development of the brain, in the metabolic process of carbohydrates, in the development of heart muscle tissue, in the development of cartilage involved in endochondral bone morphogenesis, in the differentiation of chondrocytes, in the metabolic process of chondroitin sulfate, in the morphogenesis of the embryonic skeletal system, in the endochondral ossification, in the disassembly of the extracellular matrix, in the organization of the extracellular matrix, in the process biosynthetic glycosaminoglycans, in the catabolic process of

glycosaminoglycans, in the metabolic process of glycosaminoglycans, in the metabolic process of lipoproteins, in phototransduction, visible light, protein localization, in the metabolic process of retinoids and in the metabolic process of small molecules.
5 The Nidogen, Periostin, Prolargine, s-100 protein proteins are ligand and anchor phosphorylation proteins, which are involved in the organization of the basement membrane, in the metabolic process of carbohydrates, in cell aging, cell adhesion disassembly of the cell matrix,
10 establishment of the location of proteins in the plasma membrane, organization of the extracellular matrix, metabolic process of glucosaminoglycans, biosynthetic process of keratan sulfate, catabolic process of keratan sulfate, metabolic process of keratan sulfate, membrane budding, assembly of membrane rafts , positive union regulation, positive adhesion regulation
15 from cell to substrate, positive regulation of focal adhesion assembly, positive regulation of GTPase activity, positive regulation of stress fiber assembly, positive regulation of cell dissemination dependent on substrate adhesion, protein heterotramerization, metabolic process of Small molecules, skeletal system development and tissue development.
PROTEIN ID PROTEIN NAMESNAMES OF GENESPEPTIDESSINGLE PEPTIDES
P98160 Protein protein core heparan sulfate specific basement membrane sulfateHSPG2twenty-onetwenty-one
P21810 BiglicanBGN55
P27797 CalreticulinCALR22
P02452 Alpha-1 (I) collagen chain (I)COL1A11212
P02462-2; P02462 Alpha-1 collagen chain (IV); ArrestCOL4A144
P12109 Alpha-1 collagen chain (VI)COL6A11313
P39059 Alpha-1 (XV) collagen chain;COL15A122

PROTEIN ID PROTEIN NAMESNAMES OF GENESPEPTIDESSINGLE PEPTIDES
Restina; Restina2; Restina3; Restina-4
P39060-2; P390601; P39060 Alpha-1 collagen chain (XVIII); EndostatinCOL18A122
P08123 Alpha-2 collagen chain (I)COL1A266
P08572 Alpha-2 collagen chain (IV); CanstatinCOL4A244
P12110; P12110-3; P12110-2 Alpha-2 collagen chain (VI)COL6A21010
P12111-4; P121112; P12111; P121113; P12111-5 Alpha-3 collagen chain (VI)COL6A35252
P07585; P07585-4; P07585-2; P07585-3 DecorinaDCN1010
Q12805-2; Q128054; Q12805; Q128053; Q12805-5 Fibulin-like extracellular matrix protein 1 containing EGFEFEMP199
Q07507 DermatopontinDPT44
Q9Y6C2; Q9Y6C2-2 EMILINA-1EMILIN144
P35555; P35556 Fibrillin-1FBN13131
Q9UBX5 Fibulin-5FBLN522
P02675; CON__P02676 Fibrinogen beta chain; Fibrinopeptide B; Fibrinogen beta chainFGB33
P09382 Galectin-1LGALS166
Q16363-2; Q16363 Laminin alpha-4 subunitLAMA455
P07942 Laminin beta-1 subunitLAMB133
P55268 Laminin beta-2 subunitLAMB266
P11047 Laminin gamma-1 subunitLAMC11010
P51884; CON__Q05443 LumicanoLum99
P55083; P55083-2 Microfibrile-associated glycoprotein 4MFAP422
P20774 MimecanoOGN44
P14543-2; P14543 Nidogen-1NID199

PROTEIN ID PROTEIN NAMESNAMES OF GENESPEPTIDESSINGLE PEPTIDES
Q14112; Q14112-2 Nidogen-2NID21212
Q15063-4; Q150632; Q15063-3; Q150635; Q15063; Q150637; Q150636; CON__Q2KJC7 PeriostinPOSTN66
P02545-2; P025456; P02545; P025453; P02545-5; P02545-4 Prelamina-A / C; Lamina-A / CLMNAeleveneleven
P51888 ProlarginePRELP55
P60903 S100-A10 proteinS100A1022
P22105; P22105-3; P22105-4 Tenascin-XTNXB22
Table 5: MEC and basement membrane proteins conserved in TAhd (ML4) and identified by LC-MS / MS.
5 As can be seen in the analyzes presented above, the composition of the decellularized matrices obtained is rich in all the components, which guarantees a satisfactory use in all the desired applications.
Finally, it should be added that the inventors have begun preliminary studies
10 of different material processing techniques in order to discover if the decellularized matrices of the present invention are easily susceptible to manipulation in order to produce different final products for a variety of applications. As can be seen in FIG. 8, the matrices allow their processing with various technologies: they could be dissolved and lyophilized
15 satisfactorily, micronized in liquid nitrogen, compressed and rolled to obtain a porous frame, powder, laminar and cylindrical formats that would allow the material to be perfectly applied to the applications described.
REFERENCES CITED IN THE APPLICATION
20 Gilbert TW., Et. to the. “Strategies for tissue and organ decellularization” J. Cell. Biochem 2012, vol. 113, p. 2217-2222

Crapo, PM., Et. to the. “An overview of tissue and whole processes” Biomaterials 2011, vol. 32, p. 3233-3243 organdecellularization
5 US2012 / 0264190 US2011 / 0151011
US2013 / 0202563
10 WO2011 / 132089

权利要求:
Claims (15)
[1]
one. A method of decellularization of adipose tissue comprising the steps of: a) Laminating the adipose tissue; b) Treat the adipose tissue resulting from step a) with a lipoprotein lipase: at a concentration of 10-30 u / 100 mg for 39-49 hours at 32-42 degrees Celsius or at a concentration of 45-55 u / 100 mg for 18-28 hours at 32-42 degrees Celsius; and c) Treat the adipose tissue resulting from step b) with a nuclease: at a concentration of 709-719 u / mg for 67-77 hours at 32-42 degrees Celsius or at a concentration of 1423-1433 u / mg for 35- 45 hours at 32-42 degrees Celsius or at a concentration of 1423-1433 u / mg for 67-77 hours at 32-42 degrees Celsius; so that the adipose tissue has a total DNA content equal to or less than 50 ng / mg.
[2]
2. The method of decellularization of adipose tissue according to claim 1, wherein said method does not comprise the use of organic solvents.
[3]
3. The method of decellularization of adipose tissue according to any of claims 1-2, wherein: in step b) the treatment is carried out at a concentration of 25 u / 100 mg for 44 hours at 37 degrees Celsius or one at a concentration 50 u / 100 mg for 23 hours at 37 degrees Celsius, and stage c) is performed at a concentration of 709719 u / mg for 67-77 hours at 32-42 degrees Celsius or at a concentration of 1423-1433 u / mg for 35-45 hours at 32-42 degrees Celsius or at a concentration of 1423-1433 u / mg for 67-77 hours at 32-42 degrees Celsius.

[4]
Four. The method of decellularization of adipose tissue according to any of claims 1-2, wherein: step b) is performed at a concentration of 10-30 u / 100 mg for 39-49 hours at 32-42 degrees Celsius or at concentration of 45-55 u / 100 mg for 18-28 hours at 32-42 degrees Celsius, and in stage c) the treatment is carried out at a concentration of 714 u / mg for 72 hours at 37 degrees Celsius or at a concentration of 1428 u / mg for 40 hours at 37 degrees Celsius or at a concentration of 1428 u / mg for 72 hours at 37 degrees Celsius.
[5]
5. The method of decellularizing adipose tissue according to any one of claims 1 to 4, which additionally comprises a stage between stages a) and b), wherein the adipose tissue resulting from stage a) is treated with trypsin and triton-X100.
[6]
6. The method of decellularizing adipose tissue according to any one of claims 1 to 5, further comprising a step d) comprising freezing or lyophilizing and sterilizing the adipose tissue resulting from step c).
[7]
7. The method of decellularization of adipose tissue according to any of claims 1 to 6, wherein in steps b) and c), the treatments are performed under vacuum and with stirring at 100-150 rpm.
[8]
8. The method of decellularization of adipose tissue according to any of claims 1 to 7, wherein after steps a), b) and c), there is a washing step with buffer at room temperature, under vacuum and with stirring at 100- 150 rpm, and in which in the washing stage after stage a) the buffer additionally comprises at least one antibiotic, at least one antifungal and at least one protease inhibitor, in the washing stage after stage b) the buffer comprises at least one antibiotic, at least one antifungal, at least one protease inhibitor and at least one lipase inhibitor and in which in the washing stage after step c) the buffer additionally comprises at least one antibiotic, at least one antifungal, at least one protease inhibitor and at least

a nuclease inhibitor
[9]
9. A matrix of decellularized adipose tissue obtainable by the method according to
any of claims 1 to 8.5
[10]
10. The decellularized adipose tissue according to claim 9, wherein the adipose tissue has a total DNA content of 0.015 to 0.306 ng / mg.
[11]
eleven. The decellularized adipose tissue according to any of claims 9 to
10 10, in which the adipose tissue has a total protein content of 100-931 ug / mg and a total protein content of 100-931 ug / mg and a total triglyceride content of 25-75% by weight.
[12]
12. A powder, a foam, a particle or a hydrogel comprising the decellularized adipose tissue according to any of claims 9-11.
[13]
13. A biocompatible framework or a biocompatible coating comprising the decellularized adipose tissue according to any of claims 9-11 or the powder, foam, particle or hydrogel according to claim 12.
[14]
14. The decellularized adipose tissue according to any of claims 9 to 11, the powder, the foam, the particle or the hydrogel according to claim 12, or the biocompatible framework or biocompatible coating according to claim 13 for use in tissue engineering and regenerative therapy
[15]
15. The decellularized adipose tissue for use according to claim 14, wherein the therapy is a wound healing therapy.

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同族专利:

公开号 | 公开日

ES2684856B1|2019-07-31|

WO2017114902A1|2017-07-06|

ES2684856R1|2018-10-09|

引用文献:

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

EP0821573A4|1995-04-19|2000-08-09|St Jude Medical|Matrix substrate for a viable body tissue-derived prosthesis and method for making the same|CN108079375B|2018-02-08|2021-01-15|北京桀亚莱福生物技术有限责任公司|Built-in biological sleeve and preparation method and application thereof|

CN111110919A|2019-12-30|2020-05-08|广东泓志生物科技有限公司|Preparation method of omentum majus acellular matrix material and construction method of cartilage tissue|

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优先权:

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EP15203113|2015-12-30|

PCT/EP2016/082849|WO2017114902A1|2015-12-30|2016-12-29|Method for producing a decellularized tissue matrix|

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