A method for preparing adipose decellularized matrix

Abstract

The present invention provides a method for preparing a decellularized adipose tissue matrix. Specifically, it provides a method for rapidly and efficiently preparing a decellularized adipose tissue matrix using only physical and chemical methods, without the introduction of bio-enzymes, to remove cell nuclei and lipids from allogeneic adipose tissue while preserving the matrix's natural structure. The resulting adipose tissue matrix exhibits high biosafety and contains a variety of natural bioactive components, such as collagen and proteins. It can be used in fields such as filling soft tissue volume loss caused by trauma, disease, and aging, as well as in tissue regeneration.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a method for preparing a fat decellularized matrix. Background Art

[0002] Autologous tissue transplantation is generally considered the gold standard for tissue repair, but it has certain limitations due to the limited availability of autologous material and the risk of secondary damage to the patient. The optimal scaffold for tissue repair is the extracellular matrix (ECM) of the target tissue. Commercially available allogeneic ECM products to date primarily consist of dermis, tendon, nerve, and bone tissue. Compared to xenogeneic materials, human tissue is extremely valuable and typically comes from cadaver donation programs.

[0003] During liposuction surgery, a large amount of adipose tissue is often discarded as medical waste. Studies have shown that adipose decellularized matrix extracted from fat can be used for in vitro adipose tissue engineering and in vivo adipose tissue regeneration. This is not limited to adipose tissue engineering, but also has potential applications in wound healing, bone regeneration, supporting breast tissue reconstruction, and promoting nerve repair.

[0004] The goal of tissue decellularization preparation protocols is to effectively remove all cellular and nuclear material while minimizing adverse effects on the structural composition, bioactivity, and mechanical integrity of the extracellular matrix. Decellularization protocols typically include physical, chemical, and enzymatic methods, using physical treatments or ionic solutions to dissolve cell membranes, followed by enzymatic treatment to separate cellular components from the ECM, detergents to solubilize cytoplasmic and nuclear cell components, and finally, to remove cellular debris from the tissue. After decellularization, all residual chemicals must be removed to avoid adverse reactions of the host tissue to the chemicals.

[0005] Existing methods for adipose tissue decellularization often draw on conventional tissue decellularization methods, such as the use of exogenous enzymes. Once the resulting adipose-decellularized matrix is implanted in the host, enzyme residues can impair recellularization and potentially induce an immune response. Existing methods that don't use enzymes are time-consuming, and prolonged processing can increase the spacing between collagen fibers in the matrix, alter tissue dimensions, and lead to loss of bioactive components, while also increasing the risk of bacterial growth. These long production cycles are also unfavorable for large-scale production and commercial applications.

[0006] Therefore, it is necessary to provide a new adipose decellularized matrix and its preparation method, which can quickly and effectively remove cell nuclei, lipids and viruses in adipose tissue without introducing exogenous enzymes and other biological agents, so as to solve the shortcomings of the existing technology. Summary of the Invention

[0007] The present invention aims to provide a decellularized adipose tissue matrix and its preparation method, specifically a method for rapidly and efficiently preparing a decellularized adipose tissue matrix that promotes in vivo adipocyte regeneration by removing cell nuclei and lipids using only physical and chemical methods, without the introduction of bio-enzymes. The adipose tissue matrix prepared by the present invention has high biosafety and can guide cell migration, adhesion, and differentiation. After complete degradation after injection into the body, the matrix enables the regeneration and repair of autologous adipocytes. It can be used in fields such as filling soft tissue volume loss caused by trauma, disease, and aging, as well as tissue regeneration.

[0008] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0009] The adipose decellularized matrix comprises type I collagen, type III collagen, type IV collagen, elastin, laminin, fibronectin, fatty acids, glycosaminoglycans and growth factors.

[0010] In a first aspect of the present invention, a method for preparing adipose decellularized matrix is provided, the method comprising the following steps:

[0011] S1: Cleaning

[0012] Providing a fat tissue raw material, crushing the fat tissue raw material, and rinsing it to obtain rinsed fat tissue;

[0013] S2: Pre-processing

[0014] (1) centrifuging the rinsed adipose tissue to obtain a layered mixture, removing the upper oil layer and the lower water layer, and collecting the middle layer;

[0015] (2) homogenizing the intermediate layer and performing post-processing to obtain a solid layer;

[0016] (3) rinsing the solid layer in step (2) to obtain a solid layer;

[0017] Wherein, the rinsing treatment comprises mixing the solid layer in step (2) with a degreasing agent, wherein the degreasing agent is a mixed aqueous solution of an alkaline substance, a first alcohol substance and a polyethylene glycol ether;

[0018] S3: Virus inactivation

[0019] Treating the solid layer in step (3) of step S2 with a virus inactivation reagent to obtain a solid layer;

[0020] wherein the virus inactivation treatment comprises mixing the solid layer with a virus inactivation reagent, wherein the virus inactivation reagent comprises an aqueous solution of a peroxide and a second alcohol;

[0021] S4: Decellularization

[0022] The solid layer in step S3 is treated with a decellularizing agent to obtain a fat decellularized matrix.

[0023] In another preferred embodiment, no biological enzyme is added in the method.

[0024] In another preferred embodiment, the total reaction time of the method is less than 72 hours, preferably less than 48 hours, and more preferably less than 24 hours.

[0025] In another preferred embodiment, the step S1 further comprises: allowing the adipose tissue material to stand still and removing the underlying swelling fluid.

[0026] In another preferred embodiment, in step S1, physiological saline is used for rinsing.

[0027] In another preferred embodiment, in step S1, the number of rinsing is 2-5 times, for example, 3 times.

[0028] In another preferred embodiment, the step S1 includes: standing the fat upright and removing the swelling fluid, and then rinsing with physiological saline for 3-5 times.

[0029] In another preferred embodiment, in the step (1), the centrifugal speed is 200-20000g, preferably 2000-16000g, and most preferably 8000-12000g.

[0030] In another preferred embodiment, in the step (1), the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0031] In another preferred embodiment, in step (1), the intermediate layer is a fat layer containing fat cells.

[0032] In another preferred embodiment, in the step (1), the layered mixture is divided into three layers, the upper layer is an oil layer, the middle layer is a fat layer containing fat cells, and the lower layer is a water layer.

[0033] In another preferred embodiment, the step (1) comprises: centrifuging the rinsed adipose tissue at 10,000 g for 3 minutes to obtain a layered mixture, removing the upper oil layer and the lower water layer, and collecting the middle layer (i.e., the fat layer containing adipocytes).

[0034] In another preferred embodiment, in the step (2), the homogenization is mechanical homogenization.

[0035] In another preferred embodiment, in the step (2), the homogenization treatment includes crushing and homogenizing by a homogenizer.

[0036] In another preferred embodiment, the rotation speed of the homogenizer is 5000-15000 r / min, preferably 8000-15000 r / min, such as 10000 r / min, 12000 r / min.

[0037] In another preferred embodiment, in the step (2), the homogenization treatment time is 0.5-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 1-5 min.

[0038] In another preferred embodiment, in the step (2), the post-treatment includes the steps of centrifugal separation and removal of the oil layer.

[0039] In another preferred embodiment, in the step (2), the centrifugal speed is 200-20000g, preferably 2000-16000g, and most preferably 8000-12000g.

[0040] In another preferred embodiment, in the step (2), the centrifugation time is 2-20 min, preferably 5-15 min, and more preferably 10 min.

[0041] In another preferred embodiment, the step (2) comprises: homogenizing the intermediate layer obtained in the step (1) at a speed of 10,000 r / min for 1 minute, and then centrifuging at 10,000 g to collect the solid layer.

[0042] In another preferred embodiment, in the step (3), the mixing includes oscillating mixing.

[0043] In another preferred embodiment, in the step (3), the mass ratio of the solid layer in the step (2) to the degreasing agent is 1:1 to 1:20, preferably 1:2 to 1:20, more preferably 1:5 to 1:20, for example 1:6, 1:8, 1:10.

[0044] In another preferred embodiment, the oscillation mixing time is 0.5-8 h, preferably 2-8 h, such as 3 h, 4 h.

[0045] In another preferred embodiment, the alkaline substance is selected from the group consisting of sodium hydroxide, sodium carbonate and sodium bicarbonate, or a combination thereof.

[0046] In another preferred embodiment, the first alcohol substance is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, or a combination thereof.

[0047] In another preferred embodiment, the polyethylene glycol ether is selected from the following group: fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether sodium sulfate, polyethylene glycol octylphenyl ether, or a combination thereof.

[0048] In another preferred embodiment, the concentration of the alkaline substance in the degreasing reagent is 1 mmol / L-100 mmol / L.

[0049] In another preferred embodiment, the volume fraction of the first alcohol substance in the degreasing agent is 50-100%, preferably 50-95%.

[0050] In another preferred embodiment, the volume fraction of the polyethylene glycol ether in the degreasing agent is 0.1-5%.

[0051] In another preferred embodiment, the step (3) further comprises: separating the mixture after the degreasing reagent, discarding the liquid layer, and obtaining a solid layer.

[0052] In another preferred embodiment, the separation includes centrifugal separation, or directly aspirating the liquid layer after standing, thereby discarding the liquid layer.

[0053] In another preferred embodiment, the step (3) comprises: adding a degreasing agent in a certain proportion to the solid layer in step (2), shaking and mixing for a period of time, discarding the liquid layer, and collecting the solid layer.

[0054] In another preferred embodiment, the step (3) comprises: adding an aqueous solution of sodium hydroxide, isopropyl alcohol, and polyethylene glycol octylphenyl ether in a mass ratio of 1:10 to the solid layer in step (2), shaking and mixing for 3 hours, discarding the liquid layer, and collecting the solid layer.

[0055] The preparation method for acellular matrices needs to be selected based on the specific characteristics of the target tissue, and the process should be integrated according to the cell characteristics, density, and lipid content of the tissue. Because adipose tissue contains over 80% lipid, lipid adhesion to the matrix surface reduces the efficiency of decellularization during subsequent processing. The present invention removes the vast majority of lipids from adipose tissue through a combined homogenization, centrifugation, and rinsing method, significantly reducing the time required for subsequent decellularization and improving the efficiency of adipose-decellularized matrix preparation.

[0056] In another preferred embodiment, in step S3, the mixing includes oscillating mixing.

[0057] In another preferred embodiment, in step S3, the virus inactivation treatment includes adding the solid content to a virus inactivation reagent and mixing.

[0058] In another preferred embodiment, the peroxide is selected from the group consisting of hydrogen peroxide, peracetic acid, chlorine dioxide, ozone, or a combination thereof.

[0059] In another preferred embodiment, the second alcohol substance is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, or a combination thereof.

[0060] In another preferred embodiment, the volume fraction of the peroxide in the virus inactivation reagent is 0.05-3%, preferably 0.1-2%, more preferably 0.1-1%, such as 0.5%.

[0061] In another preferred embodiment, the volume fraction of the alcohol substance in the virus inactivation reagent is 1-24%, preferably 2-20%, more preferably 2-10%, such as 4%, 5%, or 8%.

[0062] In another preferred embodiment, in the step S3, the mass ratio of the solid layer in step (3) to the virus inactivation reagent in step S2 is 1:1 to 1:20, preferably 1:2 to 1:20, more preferably 1:5 to 1:20, for example 1:8, 1:10.

[0063] In another preferred embodiment, the oscillation mixing time is 0.5-6 h, preferably 1-6 h, such as 4 h.

[0064] In another preferred embodiment, the step S3 further comprises: separating the mixture after the virus inactivation reagent, discarding the liquid layer, and then washing the remaining solid layer to obtain a solid layer.

[0065] In another preferred embodiment, the separation comprises centrifugal separation, or optionally, directly aspirating the liquid layer after standing, thereby discarding the liquid layer.

[0066] In another preferred embodiment, the washing includes: washing with PBS solution and / or water.

[0067] In another preferred embodiment, the washing includes: first washing with PBS solution by shaking, and then washing with purified water by shaking.

[0068] In another preferred embodiment, the step S3 comprises: adding the solid layer of step (3) in step S2 into a virus inactivation reagent in a certain proportion and shaking mixing, discarding the liquid layer after treatment, and washing the remaining solid with PBS solution, purified water, and sodium chloride solution in turn to obtain a solid layer.

[0069] In another preferred embodiment, the step S3 comprises: adding the solid layer of step S2 (3) into a 0.5% / 4% peracetic acid / ethanol disinfectant at a mass ratio of 1:10 and shaking mixing for 4 hours, discarding the liquid layer after treatment, and washing the remaining solid with PBS solution and purified water in turn to obtain a solid layer.

[0070] In another preferred embodiment, in step S4, the decellularization treatment includes mixing the solid contents with a decellularization reagent.

[0071] In another preferred embodiment, the mixing includes oscillating mixing.

[0072] In another preferred embodiment, in step S4, the decellularization treatment includes adding the solid contents to a decellularization reagent and mixing them.

[0073] In another preferred embodiment, in step S4, the decellularization treatment includes adding the solid contents to a decellularization reagent, mixing for a period of time, replacing the decellularization reagent with a new one, and continuing to mix.

[0074] In another preferred embodiment, the decellularization reagent is an aqueous solution of a surfactant.

[0075] In another preferred embodiment, the surfactant is selected from one or more of nonionic surfactants or ionic surfactants.

[0076] In another preferred embodiment, the surfactant is selected from one or more of SDS, Triton X-100, and sodium deoxycholate.

[0077] In another preferred embodiment, the volume fraction of the surfactant in the decellularization reagent is 0.2-5%, preferably 0.5-2%, more preferably 0.8-1.5%, for example 1%.

[0078] In another preferred embodiment, in step S4, the mass ratio of the solid layer in step S3 to the decellularization reagent is 1:1 to 1:20, preferably 1:2 to 1:20, more preferably 1:5 to 1:20, for example 1:8 or 1:10.

[0079] In another preferred embodiment, the oscillation mixing time is 1-12 h, preferably 4-12 h, such as 1 h, 2 h, 4 h, 8 h, 12 h.

[0080] In another preferred embodiment, the mixing includes first shaking treatment for t1 hour, then changing the liquid, and then shaking treatment for t2 hours.

[0081] In another preferred embodiment, t1 and t2 are each independently 2-6 h, for example 4 h.

[0082] In another preferred embodiment, the liquid replacement refers to replacing the decellularization reagent with a new one.

[0083] In another preferred embodiment, the liquid replacement includes: separation, discarding the liquid layer, and then adding new decellularization reagent.

[0084] In another preferred embodiment, the step S4 further comprises: separating the mixture treated with the decellularization reagent, discarding the liquid layer, and then washing the remaining solid layer to obtain a solid layer.

[0085] In another preferred embodiment, the separation comprises centrifugal separation, or optionally, directly aspirating the liquid layer after standing, thereby discarding the liquid layer.

[0086] In another preferred embodiment, the washing includes: washing with PBS solution and / or water.

[0087] In another preferred embodiment, the washing includes: first washing with PBS solution by shaking, and then washing with purified water by shaking.

[0088] In another preferred embodiment, the step S4 includes: adding the solid layer of step S3 into a decellularization reagent with a certain concentration in a certain proportion and shaking mixing, discarding the liquid layer after treatment, and shaking washing the remaining solid with PBS solution and purified water in turn to obtain a solid layer.

[0089] In another preferred embodiment, the step S4 includes: adding the solid layer of step S3 to 1% TritonX-100 at a mass ratio of 1:10 and shaking mixing for 8 hours, changing the liquid once every 4 hours, discarding the liquid layer after treatment, and washing the remaining solid with PBS solution and purified water in turn to obtain adipose decellularized matrix.

[0090] In another preferred embodiment, the degreasing agent treatment and the virus inactivation agent treatment optionally further include a post-treatment step of cleaning.

[0091] In another preferred embodiment, the cleaning comprises sequentially cleaning with a PBS solution and cleaning with purified water.

[0092] In a second aspect of the present invention, a fat-free cell matrix is provided. The fat-free cell matrix is prepared using the following method, which comprises the steps of:

[0093] S1: Cleaning

[0094] Providing a fat tissue raw material, crushing the fat tissue raw material, and rinsing it to obtain rinsed fat tissue;

[0095] S2: Skim

[0096] (1) centrifuging the rinsed adipose tissue to obtain a layered mixture, removing the upper oil layer and the lower water layer, and collecting the middle layer;

[0097] (2) homogenizing the intermediate layer and performing post-processing to obtain a solid layer;

[0098] (3) rinsing the solid layer in step (2) to obtain a solid layer;

[0099] Wherein, the rinsing treatment comprises mixing the solid layer in step (2) with a degreasing agent, wherein the degreasing agent is a mixed aqueous solution of an alkaline substance, a first alcohol substance and a polyethylene glycol ether;

[0100] S3: Virus inactivation

[0101] Treating the solid layer in step (3) of step S2 with a virus inactivation reagent to obtain a solid layer;

[0102] wherein the virus inactivation treatment comprises mixing the solid layer with a virus inactivation reagent, wherein the virus inactivation reagent comprises an aqueous solution of a peroxide and a second alcohol;

[0103] S4: Decellularization

[0104] The solid layer in step S3 is treated with a decellularizing agent to obtain a fat decellularized matrix.

[0105] In another preferred embodiment, the preparation method of the adipose decellularized matrix is as described in the first aspect of the present invention.

[0106] In another preferred embodiment, the adipose decellularized matrix is an allogeneic adipose decellularized matrix.

[0107] In another preferred embodiment, the adipose decellularized matrix does not contain cells and does not contain lipid droplets.

[0108] In another preferred embodiment, the lipid droplets are oil droplets released after the adipocytes are broken.

[0109] In another preferred embodiment, the “does not contain fat droplets” means that in the adipose decellularized biological material, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, and more preferably less than 0.1%.

[0110] In another preferred embodiment, the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

[0111] In another preferred embodiment, the “cell-free” refers to that the average number of cells in 1 ml of adipose decellularized biological material is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

[0112] In another preferred embodiment, the DNA content in the adipose decellularized matrix is reduced by more than 80%, preferably more than 90%, and more preferably more than 95%, compared with untreated adipocytes.

[0113] In another preferred embodiment, the adipose decellularized matrix can be homogenized to form particles.

[0114] In another preferred embodiment, the adipose decellularized matrix can be added with injection water, physiological saline and PBS solution in different proportions to prepare adipose decellularized matrix suspension.

[0115] In another preferred embodiment, the adipose decellularized matrix can be prepared into emulsions of different concentrations, which are then injection molded and freeze-dried to obtain adipose decellularized matrix sponges.

[0116] In a third aspect of the present invention, a medical material is provided, comprising: the adipose decellularized matrix according to the second aspect of the present invention, and a pharmaceutically acceptable carrier.

[0117] In another preferred embodiment, the dosage form of the medical material is a solid dosage form, a semisolid dosage form or a liquid dosage form.

[0118] In another preferred embodiment, the dosage form of the medical material includes lyophilized powder, injection, emulsion, and sponge.

[0119] In another preferred embodiment, the injection is a subcutaneous injection.

[0120] In another preferred embodiment, the lyophilized powder can be dissolved in a pharmaceutically acceptable solvent to form an injection.

[0121] In a fourth aspect of the present invention, a medical cosmetic material is provided, comprising: the adipose decellularized matrix as described in the second aspect of the present invention, and a carrier acceptable in the field of medical cosmetics.

[0122] In another preferred embodiment, the dosage form of the medical cosmetic material is a solid dosage form, a semisolid dosage form or a liquid dosage form.

[0123] In another preferred embodiment, the dosage form of the medical cosmetic material includes lyophilized powder, injection, emulsion, and sponge.

[0124] In another preferred embodiment, the injection is a subcutaneous injection.

[0125] In another preferred embodiment, the lyophilized powder can be dissolved in a pharmaceutically acceptable solvent to form an injection.

[0126] In a fifth aspect of the present invention, a tissue repair material is provided, wherein the tissue repair material comprises the adipose decellularized matrix according to the second aspect of the present invention.

[0127] In another preferred embodiment, the tissue repair material is an injectable tissue repair material.

[0128] In another preferred embodiment, the tissue repair material further comprises an acceptable carrier.

[0129] In another preferred embodiment, the tissue repair material further comprises a flow agent selected from the group consisting of physiological saline, PBS buffer, glycerol, polyethylene glycol, or a combination thereof.

[0130] In another preferred embodiment, the tissue repair material is applied to the site requiring tissue repair by injection or direct filling of a surgical opening.

[0131] In the sixth aspect of the present invention, there is provided a use of the adipose decellularized matrix as described in the second aspect of the present invention for preparing a medical material or a medical cosmetic material, wherein the medical material is used for (1) soft tissue repair and / or (2) promoting angiogenesis, and the medical cosmetic material is used for plastic surgery filling.

[0132] In another preferred embodiment, the promoting of angiogenesis includes promoting endothelial cell migration and promoting angiogenesis.

[0133] In another preferred embodiment, the soft tissue repair and / or plastic filling includes promoting cell migration and differentiation and / or promoting the growth of fat cells.

[0134] In a seventh aspect of the present invention, a method for soft tissue repair is provided, comprising administering the adipose decellularized matrix according to the second aspect of the present invention to a subject in need thereof.

[0135] In an eighth aspect of the present invention, a method for plastic surgery and filling is provided, comprising administering the adipose decellularized matrix according to the second aspect of the present invention to a subject in need thereof.

[0136] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 This is a physical picture of adipose decellularized matrix.

[0138] Figure 2 This is a photo of freeze-dried adipose decellularized matrix.

[0139] Figure 3 This is the SEM image of adipose decellularized matrix

[0140] Figure 4 This is a Masson staining image of adipose decellularized matrix.

[0141] Figure 5 This is an immunohistochemical staining of adipose decellularized matrix.

[0142] Figure 6aThis is the HE staining picture after fat tissue washing. Figure 6b This is the HE staining picture of the decellularized matrix sample.

[0143] Figure 7 These are DAPI staining images, (a) washed adipose tissue (without decellularization), and (b) decellularized matrix sample.

[0144] Figure 8 Oil Red O staining images: (a) washed adipose tissue (without decellularization), (b) decellularized matrix sample.

[0145] Figure 9 These are photos of implants taken 1 week and 4 weeks after adipose acellular matrix injection and the results of HE staining of sections.

[0146] Figure 10 Figure 3 is the HE / CD31 / Masson staining result of implant sections 4 weeks after adipose acellular matrix injection.

[0147] Figure 11 Figure 3 is the HE / CD31 / Masson staining result of implant sections 12 weeks after adipose acellular matrix injection. DETAILED DESCRIPTION

[0148] After extensive and in-depth research, the inventors have discovered for the first time a method for decellularizing adipose tissue matrix without the need for enzymes. This method, which uses only physical and chemical methods to completely remove cell nuclei and lipids, shortens the total preparation time, overcoming the drawback of prolonged preparation time without the addition of biological agents. The resulting adipose tissue matrix, when injected into the body, can promote the regeneration of adipocytes in the body. This was the basis for the present invention.

[0149] the term

[0150] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0151] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0152] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0153] Preparation method of adipose decellularized matrix

[0154] The method of the invention uses a specific degreasing agent, a virus inactivation agent and a cell removal agent, does not introduce a biological enzyme preparation, and can quickly remove cells and lipids.

[0155] In the prior art, some methods such as ball milling are used for pulverization and separation, but the collisions between the grinding balls easily introduce impurities. The present invention uses mechanical homogenization or centrifugation, which does not produce additional impurities. The present invention also adds the steps of degreasing and disinfection, which are not required in the prior art. The present invention also pre-removes lipids through a combination of homogenization, centrifugation, and rinsing to reduce the time required for subsequent decellularization and improve preparation efficiency. In addition, the present invention specifically uses a unique combination of alkali, alcohol, and polyethylene glycol ether for degreasing, and a combination of peroxide and alcohol for viral inactivation, which has excellent degreasing effect.

[0156] Adipose acellular matrix

[0157] The adipose decellularized matrix prepared by the present invention is fat-free, cell-free, and DNA-free (the removal rate of fat, cells, and DNA exceeds 90%, preferably 95%), and therefore has excellent safety. It is also rich in collagen, carbohydrates, and various cytokines, specifically IL-6, TGF-β, VEGF, BDNF, and IGF-1, and can be used as a medical material or medical cosmetic material to promote tissue repair, angiogenesis, and adipose tissue filling.

[0158] The adipose decellularized matrix of the present invention comprises type I collagen, type III collagen, type IV collagen, elastin, laminin, fibronectin, fatty acids, glycosaminoglycans and growth factors.

[0159] Specifically, the composition of the matrix sample comprises 50-80wt% (preferably 60-70wt%) of protein, 5-30wt% (preferably 10-20wt%) of collagen, 0.05-1wt% (preferably 0.1-0.8wt%) of glycosaminoglycans, 1-20pg / mL (preferably 1-5pg / mL) of IL-6, 0.05-1pg / mL (preferably 0.1-0.8pg / mL) of TGF-β, 1-20pg / mL (preferably 1-5pg / mL) of VEGF, 0.05-1pg / mL (preferably 0.1-0.8pg / mL) of BDNF, 100-500pg / mg (preferably 250-400pg / mg) of IGF-1, and 1-20wt% (preferably 1-5wt%) of fatty acids.

[0160] More specifically, the composition of the matrix sample included 62.61 wt% protein, 19.79 wt% collagen, 0.427 wt% glycosaminoglycans, 4.6 pg / mL IL-6, 0.56 pg / mL TGF-β, 2.11 pg / mL VEGF, 0.21 pg / mL BDNF, 379.31 pg / mg) of IGF-1, and 3.5 wt% fatty acids.

[0161] Medical materials and medical cosmetic materials

[0162] The present invention provides a material containing adipose decellularized matrix, which includes (but is not limited to): medical materials, medical cosmetic materials, etc.

[0163] Typically, the material is a medical material, which includes the adipose decellularized matrix according to the present invention and a pharmaceutically acceptable carrier.

[0164] In the present invention, the dosage forms of the medical material include (but are not limited to) lyophilized powder, injection, emulsion, and sponge.

[0165] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, or liquid fillers suitable for human or animal use and possessing sufficient purity and low toxicity. "Compatibility" refers to the ability of the components of a pharmaceutical composition to be compatible with the active ingredient of the drug, as well as with each other, without significantly reducing the drug's efficacy.

[0166] It should be understood that in the present invention, the carrier is not particularly limited and can be selected from materials commonly used in the art, or prepared by conventional methods, or purchased from the market. Some examples of pharmaceutically acceptable carriers include vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, chelating agents, thickeners, pH regulators, transdermal enhancers, colorants, flavorings, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0167] Typically, in addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers, and suspending agents.

[0168] The material dosage form should be compatible with the mode of administration. The material of the present invention can also be used with other synergistic therapeutic agents (including before, during or after use). When using the material of the present invention, the medicine of a safe and effective amount is applied to the desired object (such as a human or non-human mammal), and the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases is no more than about 8 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight-about 1 milligram / kg body weight. Of course, the specific dosage should also consider factors such as route of administration, patient health status, etc., and these are all within the skill range of skilled physicians.

[0169] The main advantages of the present invention include:

[0170] (1) The preparation method of the adipose decellularized matrix of the present invention can completely remove cell nuclei and lipids, and the entire process can be shortened to within 24 hours, saving production time and cost, and facilitating large-scale production.

[0171] (2) In the preparation process of the adipose decellularized matrix, the present invention only uses a combination of physical and chemical treatments, which retains the natural structure of the matrix and does not add exogenous biological enzyme preparations such as animal sources or bacterial fermentation, thereby reducing the risk of immunogens.

[0172] (3) The adipose decellularized matrix prepared by the present invention can promote the regeneration of autologous fat cells when injected into the body. The space left after the material degradation is replaced by autologous fat cells, thereby achieving the filling and regeneration of soft tissue.

[0173] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0174] The present invention prepares a fat decellularized matrix according to the following method.

[0175] Adipose tissue was obtained from six healthy women undergoing conventional liposuction (mean age 31 years (range 24-36 years)). After local anesthesia with tumescent fluid injection, fat was harvested using a 3 mm liposuction cannula with a large side hole (2 mm × 7 mm) connected to a 20 mL syringe, using artificial negative pressure to aspirate radially.

[0176] Example 1:

[0177] 1. Place the obtained fat in an upright position, remove the swelling fluid, and rinse with saline three times.

[0178] 2. The washed adipose tissue was placed in a centrifuge tube and centrifuged at 10,000 g for 3 minutes to obtain a stratified mixture. The upper oil layer and the lower water layer of the stratified mixture were removed, and the middle layer (i.e., the fat layer containing adipocytes) was collected.

[0179] The intermediate layer was homogenized at a speed of 10,000 r / min for 1 minute, and the solid layer was collected by centrifugation at 10,000 g.

[0180] An aqueous solution of sodium hydroxide, isopropyl alcohol, and polyethylene glycol octylphenyl ether was added to the solid layer at a mass ratio of 1:10, and the mixture was shaken for 3 hours, the liquid layer was discarded, and the solid layer was collected.

[0181] 3. The solid contents obtained in step 2 were added to a 0.5% / 4% peracetic acid / ethanol disinfectant at a mass ratio of 1:10 and shaken for 4 hours. The liquid layer was discarded after treatment, and the solid contents were shaken and washed with PBS solution, purified water, and sodium chloride aqueous solution to obtain the solid contents.

[0182] 4. The solid contents obtained in step 3 were added to 1% Triton X-100 at a mass ratio of 1:10 and shaken for 8 hours. The solution was changed every 4 hours. The liquid layer was discarded after treatment, and the solid contents were washed with PBS solution and purified water by shaking to obtain adipose decellularized matrix.

[0183] According to the preparation method of the embodiment, adipose decellularized matrix can be prepared within 24 hours. The obtained adipose decellularized matrix is as follows: Figure 1 As shown, the white solid substance at the bottom of the centrifuge tube is the adipose decellularized matrix. The microscopic morphology and bioactive components of the adipose decellularized matrix were measured, and the cell nucleus removal and lipid removal effects before and after decellularization of adipose tissue were measured.

[0184] Microscopic morphology: The obtained adipose decellularized matrix was placed in a -80 degree refrigerator for 12 hours and freeze-dried in a freeze dryer for 24 hours to obtain the freeze-dried adipose decellularized matrix. Figure 2 After gold spraying, the structure was observed using a scanning electron microscope. Figure 3 As shown in the figure, the microscopic morphology of the adipose decellularized matrix is a multi-layered fiber structure, and the surface of the matrix fibers is naturally wrinkled, which is conducive to cell adhesion.

[0185] Masson staining: Masson staining was performed on the matrix samples, such as Figure 4 As shown in the figure, it can be observed that the adipose decellularized matrix is almost entirely composed of blue-stained collagen with a clear fiber structure.

[0186] Immunohistochemical staining: Immunohistochemical staining was performed on the matrix samples, e.g. Figure 5 As shown in the figure, it can be observed that the adipose decellularized matrix contains a large amount of type I, type IV, type VI collagen and fibronectin, as well as elastin and laminin, which are conducive to cell adhesion, proliferation and vascular ingrowth.

[0187] Determination of cell nucleus removal: HE staining was performed on the washed adipose tissue material and the decellularized adipose decellularized matrix, respectively. Figure 6a As shown in FIG, a large number of blue-stained cell nuclei are retained in the washed material of the adipose tissue (i.e., the middle layer obtained by rinsing and centrifugation); Figure 6b As shown, no blue-stained cell nuclei were observed in the decellularized matrix sample, leaving only the red-stained matrix collagen fiber framework.

[0188] Fluorescence staining was used to establish a standard curve for residual DNA to determine residual DNA. The residual DNA in adipose tissue was reduced by more than 95% after decellularization, and the residual DNA in adipose decellularized matrix was less than 10 ng / mg.

[0189] The washed adipose tissue and the decellularized adipose decellularized matrix were stained with DAPI. Figure 7 As shown in a, under a fluorescence microscope, a large number of blue fluorescent spots of cell nuclei can be seen in the washed material of fat tissue (i.e., the middle layer obtained by rinsing and centrifugation), and as shown in Figure 7 As shown in middle b, this was not observed in the acellular matrix sample.

[0190] This indicates that the preparation method of the present invention successfully removes the cell nucleus, and the cell nucleus removal is very thorough, and no cell nucleus can be observed in the obtained decellularized matrix sample.

[0191] Lipid removal assay: The washed material of adipose tissue (i.e., the middle layer obtained by rinsing and centrifugation) and the adipose decellularized matrix after decellularization were stained with Oil Red O, as shown in Figure 5. Figure 8 As shown in a, there are a lot of lipids in the washed fat tissue (the large red parts are lipids), and Figure 8 As shown in middle b, no lipid residue was observed in the decellularized matrix sample.

[0192] Example 2

[0193] In order to better illustrate that the adipose decellularized matrix of the present invention can guide cell migration, adhesion and differentiation, and achieve the regeneration and repair of autologous fat cells, a subcutaneous injection filling test was conducted on mice using Example 1 as the subject:

[0194] Model construction: One day before modeling, shave the mice and completely expose the skin on the waist and back. Prepare a 1% sodium pentobarbital solution and inject it intraperitoneally to anesthetize the mice. Disinfect the back skin with 75% alcohol. Inject the prepared adipose decellularized matrix symmetrically into the subcutaneous tissue on both sides of the mouse's back, and give a 0.5ml graft at each injection point. When collecting the sample, first anesthetize the mouse with a 1% sodium pentobarbital solution injected intraperitoneally. After the mouse is fully anesthetized, prepare the skin and clean the injection area on the back. Use sterile scissors to cut the skin along the midline from the near-tail side of the back and free it, lift up the entire layer of the mouse skin, and separate the matrix filler. Be careful to carefully remove the fibrous connective tissue around the specimen without destroying the integrity of the specimen.

[0195] Figure 9 Adipose-derived matrix was removed one and four weeks after injection, sectioned, and stained with hematoxylin and eosin. The volume of the adipose-derived matrix changed less at four weeks than at one week, indicating good volume retention. Hematoxylin and eosin staining at one week revealed the formation of new blood vessels around the matrix implants, indicating that the matrix injection promoted endothelial cell migration and rapid formation of new blood vessels, providing an adequate blood supply for tissue regeneration. Hematoxylin and eosin staining at four weeks after injection revealed the formation of numerous adipocytes within the matrix implants.

[0196] from Figure 10 Four weeks after injection, numerous capillaries were observed surrounding the adipocytes, and multiple new adipocytes were observed surrounding the matrix material. This indicates that the injected acellular fat matrix material degrades while being replaced by new adipocytes. This demonstrates that the acellular fat matrix can guide cell migration and differentiation and promote adipocyte growth, achieving soft tissue regeneration and repair.

[0197] from Figure 11 It can be observed that 12 weeks after the injection, we use the full-thickness skin section method to more intuitively observe a large number of autologous fat cells and angiogenesis.

[0198] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for preparing adipose decellularized matrix, characterized in that: The method comprises the following steps: S1: Cleaning Providing a fat tissue raw material, crushing the fat tissue raw material, and rinsing it to obtain rinsed fat tissue; S2: Pre-processing (1) centrifuging the rinsed adipose tissue to obtain a layered mixture, removing the upper oil layer and the lower water layer, and collecting the middle layer, i.e., the fat layer containing adipocytes; (2) homogenizing the intermediate layer and performing post-processing to obtain a solid layer; the homogenizing treatment includes crushing and homogenizing the intermediate layer by a homogenizer; the post-processing includes centrifuging to remove the oil layer, and the centrifugal speed is 8000-12000g; (3) rinsing the solid layer in step (2) to obtain a solid layer; Wherein, the rinsing treatment comprises mixing the solid layer in step (2) with a degreasing agent, wherein the degreasing agent is a mixed aqueous solution of an alkaline substance, a first alcohol substance and a polyethylene glycol ether; the alkaline substance is selected from the following group: sodium hydroxide, sodium carbonate and sodium bicarbonate, or a combination thereof; the first alcohol substance is selected from the following group: methanol, ethanol, n-propanol, isopropanol, or a combination thereof; the polyethylene glycol ether is selected from the following group: fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, fatty alcohol polyoxyethylene ether sodium sulfate, polyethylene glycol octylphenyl ether, or a combination thereof; S3: Virus inactivation Treating the solid layer in step (3) of step S2 with a virus inactivation reagent to obtain a solid layer; The virus inactivation treatment comprises mixing the solid layer with a virus inactivation agent, wherein the virus inactivation agent comprises a peroxide, chlorine dioxide or ozone, and an aqueous solution of a second alcohol; the peroxide is selected from the group consisting of hydrogen peroxide, peracetic acid, or a combination thereof; the second alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, or a combination thereof; S4: Decellularization treating the solid layer in step S3 with a decellularization reagent to obtain the adipose decellularized matrix; the decellularization reagent is an aqueous solution of a surfactant, and the surfactant is selected from one or more of SDS, Triton X-100, and sodium deoxycholate; Wherein, no biological enzyme is added in the method, and the total reaction time of the method is less than 48 hours.

2. The preparation method according to claim 1, wherein The preparation method has one or more characteristics selected from the following group: In step S1 described in (a), rinsing is performed using physiological saline; (b) the alkaline substance is sodium hydroxide; the first alcohol substance is ethanol, n-propanol, isopropanol, or a combination thereof; the polyethylene glycol ether is selected from the group consisting of fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, polyethylene glycol octylphenyl ether, or a combination thereof; (c) the peroxide is selected from the group consisting of hydrogen peroxide, peracetic acid, or a combination thereof; the second alcohol is selected from the group consisting of ethanol, n-propanol, isopropanol, or a combination thereof; (d) The decellularization reagent is an aqueous solution of a surfactant; the surfactant is Triton X-100.

3. The preparation method according to claim 1, wherein The preparation method has one or more characteristics selected from the following group: In step (3) described in (a), the mass ratio of the solid layer in step (2) to the degreasing agent is 1:1 to 1:20; (b) In step S3, the mass ratio of the solid layer in step (3) to the virus inactivation reagent in step S2 is 1:1 to 1:20; (c) In step S4, the mass ratio of the solid layer in step S3 to the decellularization reagent is 1:1 to 1:

20.

4. The preparation method according to claim 2, wherein The preparation method has one or more characteristics selected from the following group: (a) the concentration of the alkaline substance in the degreasing agent is 1 mmol / L-100 mmol / L; the volume fraction of the first alcohol substance in the degreasing agent is 50-100%; the volume fraction of the polyethylene glycol ether in the degreasing agent is 0.1-5%; (b) the volume fraction of the peroxide in the virus inactivation reagent is 0.05-3%, and the volume fraction of the second alcohol substance in the virus inactivation reagent is 1-24%; (c) The volume fraction of the surfactant in the decellularization reagent is 0.2-5%.

5. The preparation method according to claim 1, wherein The preparation method has one or more characteristics selected from the following group: (a) In step (1), the centrifugal speed is 2000-16000g; In step (2) described in (b), the homogenization treatment includes crushing and homogenizing by a homogenizer; the rotation speed of the homogenizer is 8000-15000 r / min.

6. The preparation method according to claim 1, wherein The step S1 comprises: placing the fat in an upright position, removing the swelling fluid, and then rinsing with physiological saline for 3-5 times; The step (3) comprises: adding a degreasing agent in a certain proportion to the solid layer in step (2), shaking and mixing for a period of time, discarding the liquid layer, and collecting the solid layer; The step S3 comprises: adding the solid layer of step S2 (3) into a virus inactivation reagent in a certain proportion and shaking mixing, discarding the liquid layer after treatment, and washing the remaining solid with PBS solution and purified water in turn to obtain a solid layer; The step S4 comprises: adding the solid layer of step S3 into a decellularization reagent at a certain concentration in a certain proportion and shaking mixing, discarding the liquid layer after treatment, and washing the remaining solid with PBS solution and purified water in turn to obtain a solid layer.

7. The preparation method according to claim 1, wherein The preparation method has one or more characteristics selected from the following group: In step (3) described in (a), the mixing includes oscillating mixing, and the oscillating mixing time is 0.5-8h; (b) In step S3, the mixing includes oscillating mixing, and the oscillating mixing time is 0.5-6h; (c) In step S4, the decellularization treatment includes adding the solid contents to the decellularization reagent, mixing for a period of time, replacing the decellularization reagent with a new one, and continuing to mix. The mixing includes first shaking treatment for t1 hour, then changing the liquid, and then shaking treatment for t2 hours, wherein t1 and t2 are each independently 2-6 hours.

8. A fat-free cell matrix, characterized in that: The adipose decellularized matrix is prepared using the preparation method according to claim 1.

9. The adipose decellularized matrix according to claim 8, wherein The adipose decellularized matrix does not contain cells and does not contain lipid droplets; The term "free of fat droplets" means that the volume of oil droplets in the adipose decellularized biomaterial accounts for less than 1% of the total liquid, and the term "free of cells" means that the average number of cells in 1 ml of adipose decellularized biomaterial is ≤1.

10. A medical material, comprising: The adipose decellularized matrix according to claim 8, and a pharmaceutically acceptable carrier.

11. The medical material according to claim 10, wherein The dosage forms of the medical material include freeze-dried powder, injection, emulsion and sponge.

12. A medical cosmetic material, comprising: The adipose decellularized matrix according to claim 8, and an acceptable carrier in the field of medical cosmetology.

13. The medical cosmetic material according to claim 12, wherein: The dosage forms of the medical cosmetic material include freeze-dried powder, injection, emulsion, and sponge.

14. Use of the adipose decellularized matrix according to claim 8 for preparing a medical material or a medical cosmetic material, wherein the medical material is used for (1) soft tissue repair and / or (2) promoting angiogenesis, and the medical cosmetic material is used for plastic surgery and filling.

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