A method of modifying a decellularized adipose matrix with low immunogenicity

Abstract

The application relates to the technical field of biomedical materials, and discloses a method for modifying a low-immunogenicity decellularized fat matrix. The method comprises the following steps: washing and crushing fat tissue raw materials to obtain rinsed fat tissue; centrifugally separating the rinsed fat tissue, collecting a fat layer, and then uniformly homogenizing and post-treating the fat layer to obtain a solid layer after defatting; mixing the solid layer after defatting with a virus inactivation reagent to perform virus inactivation; mixing the solid layer after virus inactivation with a decellularization reagent to perform decellularization treatment, and obtaining a decellularized fat matrix; and finally performing low-immunogenicity modification treatment on the decellularized fat matrix to obtain a low-immunogenicity decellularized fat matrix. The combination of centrifugal separation and homogenization treatment realizes efficient defatting, the subsequent virus inactivation, decellularization and modification steps are synergistically enhanced, the immunogenicity of the matrix can be effectively reduced, the extracellular matrix structure and active ingredients are well reserved, the operation is simple, and the method is suitable for various fat sources.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically a method for modifying a decellularized adipose matrix with low immunogenicity. Background Technology

[0002] Acellular adipose-derived matrix is ​​a type of extracellular matrix scaffold material made from adipose tissue after decellularization to remove cellular components. Rich in collagen, elastin, mucopolysaccharides, and various growth factors, it possesses excellent biocompatibility and a porous three-dimensional structure, making it promising for applications in soft tissue repair, wound healing, breast reconstruction, and adipose tissue engineering. However, adipose-derived matrices still face the problem of residual immunogenicity in clinical applications. Residual DNA, α-Gal antigens, and various intracellular proteins after decellularization may still induce host immune responses, leading to graft absorption, fibrosis, or even rejection, thus limiting its widespread application in allogeneic or xenograft transplantation.

[0003] Currently, several methods for preparing decellularized adipose matrix have been reported. The standard procedure includes steps such as cleaning, defatting, decellularization, washing, and sterilization of adipose tissue. Defatting often involves immersion extraction with organic solvents such as isopropanol, acetone, or a chloroform-methanol mixture. While this effectively removes lipids, it carries a high risk of organic solvent residue, and repeated immersion can damage the ultrastructure and mechanical properties of the extracellular matrix. Decellularization methods primarily rely on chemical reagents such as trypsin-EDTA, SDS (sodium dodecyl sulfate), Triton X-100, or sodium deoxycholate to achieve cell removal by disrupting cell membranes and dissolving nucleoproteins. While these methods can reduce DNA content to some extent, they share the following common problems: First, strong chemical detergents such as SDS, while efficiently decellularizing, can excessively strip glycosaminoglycans and growth factors from the matrix, weakening the scaffold's bioactivity. Second, excessively long digestion times or high concentrations of trypsin can degrade collagen and elastic fibers, impairing the scaffold's mechanical strength and resistance to degradation. Third, the processing efficiency of single decellularization reagents is limited, often requiring extended processing times or increased concentrations, which further exacerbates damage to matrix components.

[0004] For immunogenic modification, existing techniques often involve additional processing steps after decellularization, such as using α-galactosidase to remove Gal antigens, using nucleases to degrade residual DNA, or using cross-linking agents like glutaraldehyde to block antigenic epitopes. However, α-galactosidase is expensive and has poor enzyme activity stability, nuclease treatment requires specific buffer systems and temperature conditions, and is cumbersome to operate, while glutaraldehyde cross-linking, although it can reduce immunogenicity, is prone to causing cytotoxicity and altering matrix degradation characteristics. In addition, the above modification steps are usually performed independently after all decellularization is completed, which not only prolongs the preparation cycle but also increases the risk of intermediate product contamination.

[0005] It is worth noting that the processing order of adipose tissue itself has a significant impact on decellularization efficiency and matrix retention. Most existing methods first chop and rinse the adipose tissue, then directly perform defatting (soaking in organic solvents) or proceed directly to the decellularization step, without fully considering the synergistic effect of centrifugation and homogenization. Adipose tissue contains a large number of mature adipocytes, whose intracellular spaces are filled with lipid droplets, and whose cell membranes are fragile and easily ruptured. If proper centrifugation and controlled homogenization are not performed before defatting, the released free lipids will encapsulate matrix fragments, hindering the contact between the decellularization reagent and the matrix, leading to uneven decellularization. On the other hand, the position of the virus inactivation step in the decellularization process is also crucial. If virus inactivation is performed before decellularization, the oxidative properties of the inactivation reagent may exacerbate matrix protein denaturation; if it is performed after decellularization, residual cell debris may protect virus particles from inactivation, reducing the inactivation effect. Summary of the Invention

[0006] The purpose of this invention is to provide a method for modifying acellular adipose matrix with low immunogenicity, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides a method for modifying acellular adipose matrix with low immunogenicity, the method comprising:

[0008] S1: The raw adipose tissue is cleaned and crushed to obtain rinsed adipose tissue;

[0009] S2: The rinsed adipose tissue is pretreated, the pretreatment including centrifugation, collection of the fat layer, homogenization and posttreatment, to obtain a defatted solid layer;

[0010] S3: Perform virus inactivation treatment on the degreased solid layer, wherein the virus inactivation treatment includes mixing the degreased solid layer with a virus inactivation reagent to obtain a virus-inactivated solid layer.

[0011] S4: Decellularize the solid layer after virus inactivation, wherein the decellularization process includes mixing the solid layer after virus inactivation with a decellularization reagent to obtain a decellularized lipid matrix;

[0012] S5: The decellularized adipose matrix is ​​subjected to low immunogenicity modification treatment to obtain a low immunogenicity decellularized adipose matrix.

[0013] Preferably, in step S1, the cleaning and crushing of the adipose tissue raw material includes:

[0014] After the adipose tissue material is allowed to stand to remove the lower layer of swelling fluid, it is rinsed with physiological saline 2-5 times, and then the rinsed adipose tissue material is mechanically crushed to obtain rinsed adipose tissue.

[0015] Preferably, in step S2, the centrifugation and collection of the fat layer includes:

[0016] The rinsed adipose tissue was centrifuged at 8000-12000g for 1-5 minutes to obtain a mixture with a three-layer structure, wherein 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.

[0017] Remove the upper oil layer and the lower water layer, and collect the intermediate layer.

[0018] Preferably, in step S2, the homogenization and post-processing include:

[0019] The intermediate layer is mechanically homogenized at a speed of 8000-15000 r / min for 1-5 minutes to obtain a homogenized product;

[0020] The homogenized product is centrifuged at 8000-12000g for 5-15 minutes to remove the upper oil layer and collect the lower solid layer.

[0021] Preferably, in step S2, obtaining the degreased solid layer further includes:

[0022] The lower solid layer is mixed with a degreasing agent at a mass ratio of 1:5 to 1:20 and treated under shaking conditions for 2-8 hours. The degreasing agent is an aqueous solution containing 1-100 mmol / L of alkaline substance, 50-95% by volume of a first alcohol substance, and 0.1-5% by volume of polyethylene glycol ether.

[0023] The treated mixture is centrifuged or allowed to stand before the liquid layer is removed and the solid layer is collected to obtain the degreased solid layer.

[0024] Preferably, in step S3, mixing the defatted solid layer with the virus inactivation reagent includes:

[0025] The defatted solid layer is mixed with the virus inactivation reagent at a mass ratio of 1:5 to 1:20 and treated under shaking conditions for 1-6 hours. The virus inactivation reagent is an aqueous solution containing 0.1-2% peroxide and 2-10% second alcohol by volume.

[0026] The treated mixture is centrifuged or allowed to stand before the liquid layer is removed and the solid layer is collected.

[0027] The solid layer was washed sequentially with PBS solution and purified water by shaking to obtain the virus-inactivated solid layer.

[0028] Preferably, in step S4, mixing the virus-inactivated solid layer with the decellularization reagent includes:

[0029] The solid layer after virus inactivation is mixed with the decellularization reagent at a mass ratio of 1:5 to 1:20 and treated under shaking conditions for 4-12 hours. The decellularization reagent is an aqueous solution containing 0.5-2% by volume of a surfactant, which is selected from one or more of SDS, Triton X-100 and sodium deoxycholate.

[0030] During the process, the decellularization reagent should be replaced every 2-6 hours;

[0031] The treated mixture is centrifuged or allowed to stand before the liquid layer is removed and the solid layer is collected.

[0032] The solid layer was washed sequentially with PBS solution and purified water by shaking to obtain the decellularized lipid matrix.

[0033] Preferably, in step S5, the modifying agent is an aqueous solution containing 0.5-5% by volume fatty alcohol polyoxyethylene ether and 1-10% by volume a third alcohol.

[0034] The decellularized lipid matrix and the modifying agent were mixed at a mass ratio of 1:5 to 1:15 and reacted with shaking at 25-37°C for 6-18 hours.

[0035] The mixture after the reaction was centrifuged or allowed to stand before the liquid layer was removed and the solid layer was collected. The solid layer was then washed with PBS solution and purified water in sequence to obtain the low-immunogenic decellularized lipid matrix.

[0036] Preferably, in step S5, the modifying reagent is a MES buffer containing 0.1-2% by mass of N-hydroxysuccinimide and 0.1-1% by mass of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, wherein the concentration of the MES buffer is 10-100 mM and the pH is 5.0-6.5.

[0037] The decellularized lipid matrix and the modifying agent were mixed at a mass ratio of 1:8 to 1:20 and reacted with shaking at 20-30°C for 2-12 hours.

[0038] The mixture after the reaction was centrifuged or allowed to stand before the liquid layer was removed and the solid layer was collected. The solid layer was then washed with PBS solution and purified water in sequence to obtain the low-immunogenic decellularized lipid matrix.

[0039] Preferably, in step S5, the modifying reagent is a phosphate buffer containing 0.1-1% glutaraldehyde and 1-5% a fourth alcohol by volume, wherein the concentration of the phosphate buffer is 10-50 mM and the pH is 7.2-7.8.

[0040] The decellularized lipid matrix and the modifying agent were mixed at a mass ratio of 1:10 to 1:20 and reacted with shaking at 25-37°C for 1-8 hours.

[0041] The mixture after the reaction was centrifuged or allowed to stand before the liquid layer was removed and the solid layer was collected. The solid layer was then washed with PBS solution and purified water in sequence to obtain the low-immunogenic decellularized lipid matrix.

[0042] Compared with the prior art, the beneficial effects of the present invention are:

[0043] The method for modifying adipose tissue matrix with low immunogenicity provided by this invention integrates the washing, crushing, centrifugation, homogenization, virus inactivation, decellularization, and low immunogenicity modification of adipose tissue raw materials in a rational and orderly manner, forming a coherent and synergistic preparation process. The method first washes and crushes the adipose tissue raw materials to remove residual swelling fluid and blood components, providing a homogeneous initial material for subsequent centrifugation. Then, centrifugation at specific speeds and times allows the adipose tissue to naturally separate into three layers. By precisely collecting the middle layer containing adipocytes and removing the oil and water layers, interference from free lipids and water-soluble impurities is effectively eliminated, resulting in a purer homogenization process. This reduces the lubricating and hindering effect of lipids on the homogenization process, allowing mechanical homogenization to more fully break down adipocytes and release intracellular lipid droplets while preserving the fibrous network structure of the extracellular matrix. After homogenization, the mixture is centrifuged again at a high speed to remove the upper oil layer and collect the lower solid layer. This operation separates the lipids released from the homogenate in a timely manner, preventing the lipids from re-adsorbing onto the matrix surface during subsequent virus inactivation and decellularization. This ensures that the virus inactivation reagent and the decellularization reagent can directly and uniformly contact the matrix components, thereby achieving efficient virus inactivation and cell clearance with lower reagent concentrations and shorter processing times, and reducing excessive damage to the active components of the matrix.

[0044] In the virus inactivation step, this invention directly mixes the defatted solid layer with the virus inactivation reagent. Since this solid layer has undergone two rounds of centrifugation and homogenization, its structure is loose, flocculent, or particulate, significantly increasing its specific surface area. This allows the virus inactivation reagent to rapidly penetrate the matrix, achieving uniform and thorough virus inactivation while avoiding incomplete inactivation due to the lipid barrier, thus improving biosafety. The solid layer obtained after virus inactivation is then mixed with a decellularization reagent for decellularization. The decellularization reagent, also benefiting from the matrix's high permeability, works rapidly, effectively lysing residual cell membranes and degrading nucleic acid components. Furthermore, since most lipids have been removed in the preceding steps, the decellularization reagent is not consumed or emulsified by lipids, maintaining its effective concentration. This reduces the amount of decellularization reagent used and the reaction time, lowering the risk of reagent residue.

[0045] This invention arranges the low immunogenicity modification treatment after decellularization. However, the decellularization process itself, through a combination of centrifugation-homogenization-centrifugation, has already resulted in a highly porous and exposed matrix. Therefore, the modification reagent (e.g., amino-containing biocompatible macromolecules or antigen blocking agents) can efficiently bind to the exposed antigenic epitopes in the matrix, achieving in-situ modification. This modification step is compatible with the decellularization step in terms of reaction conditions, allowing direct modification using the washing buffer system after decellularization. It eliminates the need to change extreme pH or temperature conditions or add lengthy dialysis or replacement steps, simplifying the process and shortening the overall preparation time. Simultaneously, because decellularization significantly reduces the content of DNA and soluble proteins, the main targets of the modification reagent (i.e., antigenic determinants on residual insoluble structural proteins) are more concentrated, improving modification efficiency and increasing the number of modification groups bound per unit mass of matrix. This allows for satisfactory immunogenicity shielding with a lower reagent dosage ratio, reducing reagent waste and potential cytotoxicity. Attached Figure Description

[0046] Figure 1 This diagram illustrates the operational steps of a method for modifying a decellularized adipose matrix with low immunogenicity according to the present invention. Detailed Implementation

[0047] This invention discloses a method for modifying adipose tissue matrix with low immunogenicity. The core process includes five main steps: adipose tissue pretreatment, defatting treatment, virus inactivation, decellularization treatment, and low immunogenicity modification. By optimizing the process parameters, reagent formulations, and reaction conditions of each step, cell debris, nucleic acids, lipids, and viral contaminants in adipose tissue are effectively removed. At the same time, the decellularized adipose tissue matrix is ​​specifically modified, significantly reducing the immunogenicity of the matrix while retaining its natural three-dimensional porous structure and biocompatibility. This method can be widely used in soft tissue repair, tissue engineering scaffolds, regenerative medicine, and other fields.

[0048] The raw materials and reagents used in the following examples and comparative examples are all commercially available conventional biological experimental reagents. The experimental environment is a sterile ultra-clean workbench with an ambient temperature of 25°C and a relative humidity of 55%. All centrifugation, shaking, and cleaning operations follow aseptic biological experimental standards to ensure the accuracy and repeatability of experimental data.

[0049] Example 1

[0050] See appendix Figure 1 This invention provides a method for modifying acellular adipose matrix with low immunogenicity, comprising the following steps:

[0051] S1. Raw material cleaning and crushing: Fresh human liposuction adipose tissue is selected as raw material. The adipose tissue raw material is left to stand at room temperature for 15 minutes to thoroughly remove the lower layer of turbid swelling fluid. The adipose tissue raw material is rinsed 3 times with sterile saline, with each rinse lasting 5 minutes, to remove residual blood, swelling fluid and impurities. After rinsing, the adipose tissue is mechanically crushed at low temperature using a sterile mechanical crusher at a crushing speed of 5000 r / min for 2 minutes to obtain uniform, fine, and granular adipose tissue.

[0052] S2. Pretreatment to prepare the defatted solid layer: The rinsed adipose tissue obtained in S1 was placed in a sterile centrifuge tube and centrifuged at 10000g for 3 minutes. After centrifugation, the mixture formed a three-layer structure: an upper oil layer, a middle layer rich in adipocytes, and a lower aqueous impurity layer. Under sterile conditions, the upper oil layer and the lower aqueous layer were removed, and the middle fat layer was accurately collected. The collected middle fat layer was placed in a sterile homogenizer and mechanically homogenized at 12000r / min for 3 minutes to obtain a homogenized homogenate. The homogenate was centrifuged at 10000g for 10 minutes, and the upper oil layer was removed, collecting the lower solid layer. The lower solid layer was mixed with a defatting reagent at a mass ratio of 1:10 and placed in a constant temperature shaking incubator, where it was continuously shaken at a shaking rate of 150r / min for 5 hours. The defatting reagent was a sterile aqueous solution containing 50mmol / L sodium hydroxide, 75% ethanol (v / v), and 2% polyethylene glycol ether (v / v). After processing, centrifuge at 8000g for 8 minutes, remove the upper liquid layer, and collect the bottom solid layer, which is the degreased solid layer.

[0053] S3. Virus inactivation treatment: The defatted solid layer obtained in S2 was mixed with the virus inactivation reagent at a mass ratio of 1:10 and treated with constant temperature shaking at 150 r / min for 3 h; the virus inactivation reagent was a sterile aqueous solution containing 1% hydrogen peroxide and 6% ethanol by volume. After the reaction, the mixture was allowed to stand and separate into layers. The upper layer of inactivation waste liquid was aspirated off, and the bottom solid layer was collected. The solid layer was then washed sequentially with sterile PBS solution and sterile purified water by shaking, with each wash lasting 3 min and a washing speed of 120 r / min, for a total of 3 washes each time, to thoroughly remove residual inactivation reagent. After drying, the virus-inactivated solid layer was obtained.

[0054] S4. Decellularization: The virus-inactivated solid layer obtained in S3 was mixed with decellularization reagent at a mass ratio of 1:10 and placed in a constant temperature shaking incubator at 150 rpm for 8 hours. The decellularization reagent was a sterile aqueous solution containing 1% Triton X-100 (v / v). During the decellularization process, the decellularization reagent was replaced with fresh reagent every 4 hours to ensure the decellularization reaction proceeded fully. After treatment, the layers were allowed to separate, the upper decellularization waste liquid was aspirated, and the solid layer was collected. The solid layer was then washed 4 times with sterile PBS solution and 3 times with sterile purified water, each wash lasting 5 minutes at a speed of 120 rpm, to remove residual decellularization reagent and cell debris, obtaining a pure decellularized lipid matrix.

[0055] S5. Low Immunogenicity Modification Treatment: This example uses a fatty alcohol polyoxyethylene ether-alcohol modification system. The modification reagent is prepared as a sterile aqueous solution containing 2% (v / v) fatty alcohol polyoxyethylene ether and 5% (v / v) ethanol. The decellularized lipid matrix obtained in S4 is mixed with the modification reagent at a mass ratio of 1:10 and placed in a constant temperature environment at 30℃ with shaking at 150 rpm for 12 h. After the reaction, the mixture is allowed to stand and separate into layers. The upper layer of modification waste liquid is aspirated, and the bottom solid layer is collected. The mixture is then washed sequentially with sterile PBS solution and sterile purified water with shaking, 4 times each, with each wash lasting 4 min, to thoroughly remove residual modification reagent and finally obtain a low immunogenicity decellularized lipid matrix.

[0056] Example 2

[0057] A method for modifying acellular adipose matrix with low immunogenicity includes the following steps:

[0058] S1. Raw material cleaning and crushing: Fresh human liposuction fat tissue is selected as raw material and left to stand at room temperature for 20 minutes to remove the lower swelling fluid; it is rinsed 4 times with sterile saline, each rinse lasting 4 minutes, to remove impurities and residual waste liquid; it is crushed for 3 minutes at 5000 r / min using a low-temperature mechanical crusher to obtain the rinsed fat tissue.

[0059] S2. Pretreatment to prepare the defatted solid layer: The rinsed adipose tissue was centrifuged at 12000g for 2 min, and after separation, the upper oil layer and the lower water layer were removed, and the middle fat layer was collected. The middle fat layer was homogenized at 15000r / min for 2 min to obtain a homogenate. The homogenate was centrifuged at 12000g for 5 min to remove the upper oil layer, and the lower solid layer was collected. The lower solid layer was mixed with a defatting reagent at a mass ratio of 1:15 and treated with shaking at 150r / min for 6 h. The defatting reagent was an aqueous solution containing 80mmol / L potassium hydroxide, 85% isopropanol (v / v), and 3% polyethylene glycol ether (v / v). After treatment, the mixture was centrifuged at 9000g for 6 min, the liquid layer was removed, and the solid layer was collected to obtain the defatted solid layer.

[0060] S3. Virus inactivation treatment: The defatted solid layer was mixed with the virus inactivation reagent at a mass ratio of 1:15 and shaken at 150 rpm for 4 hours. The virus inactivation reagent was an aqueous solution containing 1.5% peracetic acid and 8% isopropanol. After the reaction, the mixture was allowed to stand and separate into layers. The waste liquid was removed, and the solid layer was collected. The solid layer was then washed sequentially with PBS solution and purified water, three times each, for three minutes each time, to obtain the virus-inactivated solid layer.

[0061] S4. Decellularization: The virus-inactivated solid layer was mixed with a decellularization reagent at a mass ratio of 1:15 and treated with shaking at 150 rpm for 10 h. The decellularization reagent was a sterile aqueous solution containing 1.5% SDS by volume, and was replaced every 5 h during the treatment. After the reaction, the mixture was allowed to stand and the liquid was removed. The solid layer was collected and washed 4 times with PBS solution and 3 times with purified water, each wash lasting 5 min, to obtain the decellularized lipid matrix.

[0062] S5. Low Immunogenicity Modification Treatment: In this example, a carbodiimide cross-linking modification system was used. The modification reagent was prepared as follows: a 50 mM MES buffer solution with pH 5.8, containing 0.8% N-hydroxysuccinimide and 0.5% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The decellularized lipid matrix and the modification reagent were mixed at a mass ratio of 1:12 and reacted at 25°C with shaking at 150 rpm for 8 hours. After the reaction, the mixture was allowed to stand and separate into layers. The upper waste liquid was removed, and the solid layer was collected. The matrix was then washed four times each with PBS solution and purified water, each wash lasting 4 minutes, to obtain a low immunogenic decellularized lipid matrix.

[0063] Example 3

[0064] A method for modifying acellular adipose matrix with low immunogenicity includes the following steps:

[0065] S1. Raw material cleaning and crushing: Select fresh human liposuction fat tissue, let it stand at room temperature for 10 minutes to remove swelling fluid; rinse twice with physiological saline, each rinse for 6 minutes; crush with a mechanical crusher at 4500 r / min for 4 minutes to obtain rinsed fat tissue.

[0066] S2. Pretreatment to prepare the defatted solid layer: The rinsed adipose tissue was centrifuged at 8000g for 5 min, and after separation, the oil and water layers were removed, and the middle fat layer was collected. The middle fat layer was homogenized at 8000r / min for 5 min to obtain a homogenate product. The homogenate product was centrifuged at 8000g for 15 min, the oil layer was removed, and the lower solid layer was collected. The solid layer was mixed with a defatting reagent at a mass ratio of 1:5 and shaken at 120r / min for 2 h. The defatting reagent was an aqueous solution containing 10mmol / L sodium hydroxide, 50% ethanol (v / v), and 0.1% polyethylene glycol ether (v / v). The mixture was centrifuged at 10000g for 5 min, the liquid was removed, and the defatted solid layer was collected.

[0067] S3. Virus inactivation treatment: The defatted solid layer was mixed with the virus inactivation reagent at a mass ratio of 1:5 and shaken at 120 rpm for 1 hour. The virus inactivation reagent was an aqueous solution containing 0.1% hydrogen peroxide and 2% ethanol by volume. After standing and removing the liquid, the mixture was washed twice each with PBS and purified water to obtain the virus-inactivated solid layer.

[0068] S4. Decellularization: The virus-inactivated solid layer and decellularization reagent were mixed at a mass ratio of 1:5 and shaken at 120 rpm for 4 hours. The decellularization reagent was an aqueous solution containing 0.5% sodium deoxycholate by volume, and the reagent was changed every 2 hours. After standing and removing the liquid, the mixture was washed sequentially with PBS and purified water to obtain the decellularized lipid matrix.

[0069] S5. Low Immunogenicity Modification Treatment: In this example, a glutaraldehyde cross-linking modification system was used. The modification reagent was prepared as follows: a 30 mM phosphate buffer solution with a pH of 7.4, containing 0.5% glutaraldehyde and 3% butanol (v / v). The decellularized adipose matrix was mixed with the modification reagent at a mass ratio of 1:10 and reacted at 25°C and 120 rpm for 4 hours. After the reaction, the waste liquid was removed, and the solid layer was thoroughly washed with PBS and purified water to obtain a low-immunogenic decellularized adipose matrix.

[0070] Example 4

[0071] A method for modifying acellular adipose matrix with low immunogenicity includes the following steps:

[0072] S1. Raw material cleaning and crushing: Fresh human liposuction adipose tissue is left to stand at room temperature for 20 minutes to remove swelling fluid, rinsed 5 times with physiological saline, each rinse lasting 3 minutes; then mechanically crushed at low temperature at 6000 r / min for 2 minutes to obtain rinsed adipose tissue.

[0073] S2. Pretreatment to prepare the defatted solid layer: After rinsing the adipose tissue, centrifuge at 12000g for 1 min, and collect the intermediate fat layer after separation. Homogenize the intermediate fat layer at 15000r / min for 1 min, and centrifuge the homogenate at 12000g for 5 min to remove the oil layer and collect the solid layer. Mix the solid layer with the defatting reagent at a mass ratio of 1:20 and shake at 180r / min for 8 h. The defatting reagent is an aqueous solution containing 100mmol / L potassium hydroxide, 95% isopropanol (v / v), and 5% polyethylene glycol ether (v / v). Centrifuge at 8000g for 10 min to remove the liquid and collect the defatted solid layer.

[0074] S3. Virus inactivation treatment: The defatted solid layer was mixed with the virus inactivation reagent at a mass ratio of 1:20 and shaken at 180 r / min for 6 h. The virus inactivation reagent was an aqueous solution containing 2% peracetic acid and 10% isopropanol by volume. After standing and separating the layers, the waste liquid was removed, and the mixture was washed 4 times each with PBS and purified water to obtain the virus-inactivated solid layer.

[0075] S4. Decellularization: The virus-inactivated solid layer and decellularization reagent were mixed at a mass ratio of 1:20 and shaken at 180 rpm for 12 h. The decellularization reagent was a 2% Triton X-100 aqueous solution, which was replaced every 6 h. After the reaction, the solution was washed to obtain a high-purity decellularized lipid matrix.

[0076] S5. Low Immunogenicity Modification Treatment: A composite modification system was used, with the modifying reagent being an aqueous solution of 5% (v / v) fatty alcohol polyoxyethylene ether and 10% (v / v) ethanol. The decellularized matrix and the modifying reagent were mixed at a mass ratio of 1:15 and reacted at 37℃ and 180 rpm for 18 h with shaking. After thorough washing to remove residual reagents, a low immunogenic decellularized fatty matrix was obtained.

[0077] Comparative Example 1

[0078] The process of this comparative example is basically the same as that of Example 1, except that the S5 low immunogenicity modification step is omitted, and the decellularized lipid matrix prepared in S4 is directly used as the final product. The remaining process parameters, reagent formulations, and operating steps are exactly the same. The specific steps are as follows:

[0079] S1. Raw material cleaning and crushing: exactly the same as in Example 1, to obtain rinsed adipose tissue.

[0080] S2. Preparation of the degreased solid layer: The process is exactly the same as in Example 1, and a degreased solid layer is obtained.

[0081] S3. Virus inactivation treatment: exactly the same as in Example 1, to obtain a solid layer after virus inactivation.

[0082] S4. Decellularization treatment: Completely consistent with Example 1, to obtain a decellularized lipid matrix, and use the unmodified decellularized lipid matrix as the final sample.

[0083] Comparative Example 2

[0084] This comparative example is basically the same as the process in Example 1, except that the S2 degreasing step is omitted. All other process parameters, reagent formulations, and operating steps are identical. The specific steps are as follows:

[0085] S1. Raw material cleaning and crushing: exactly the same as in Example 1, to obtain rinsed adipose tissue.

[0086] S2. Pretreatment: Only centrifugation to separate layers, collection of fat layer, homogenization, and centrifugation to remove oil are performed. No degreasing reagent is added for degreasing treatment. The lower solid layer is directly collected for subsequent steps.

[0087] S3. Virus inactivation treatment: completely consistent with Example 1.

[0088] S4. Decellularization treatment: Completely consistent with Example 1.

[0089] S5. Low immunogenicity modification treatment: Completely consistent with Example 1, to obtain a modified decellularized lipid matrix that is not completely defat-free.

[0090] Comparative Example 3

[0091] This comparative example is basically the same as the process in Example 2, except that: the decellularization reagent is not changed during the S4 decellularization process, and an equal amount of decellularization reagent is used to complete the reaction in one go. The other process parameters, reagent formulations, and operating steps are exactly the same. The specific steps are as follows:

[0092] S1. Raw material cleaning and crushing: completely consistent with Example 2.

[0093] S2. Pretreatment to prepare the degreased solid layer: completely consistent with Example 2.

[0094] S3. Virus inactivation treatment: completely consistent with Example 2.

[0095] S4. Decellularization: The reagent ratio, reaction temperature, oscillation rate, and total reaction time are the same as in Example 2. The difference is that the decellularization reagent is not changed throughout the 10-hour decellularization reaction. After the reaction is completed, the solid layer is washed and collected.

[0096] S5. Low immunogenicity modification treatment: Completely consistent with Example 2, to obtain the corresponding matrix sample.

[0097] The decellularized lipid matrices prepared in each embodiment and comparative example were subjected to core performance tests, including: cell residue rate, DNA residue, lipid residue, immunogenicity score, and pore integrity rate. Each test was performed in triplicate, and the average value was used as the final result to ensure data objectivity.

[0098] 1. Cell residue rate: The matrix sections were observed using the hematoxylin-eosin (HE) staining method, the number of residual cells in the field of view was counted, and the cell residue rate was calculated.

[0099] 2. DNA Residual Content: Residual DNA in the matrix was extracted using a genomic DNA extraction kit, and the DNA content was determined by ultraviolet spectrophotometer. The standard is that the residual DNA in the biomedical matrix is ​​<50 ng / mg.

[0100] 3. Lipid Residual Content: The residual lipid content in the matrix was determined using the Soxhlet extraction method to evaluate the defatting effect;

[0101] 4. Immunogenicity score: The secretion of inflammatory factors TNF-α and IL-6 was detected by co-culturing mouse macrophages RAW264.7 and combined with cell proliferation rejection reaction, and scored from 1 to 10. The lower the score, the lower the immunogenicity.

[0102] 5. Pore integrity rate: The three-dimensional pore structure of the matrix is ​​observed by scanning electron microscopy, the proportion of intact pores is counted, and the integrity of the matrix structure is evaluated.

[0103] Table 1: Comparison of matrix decellularization and degreasing purification performance between each embodiment and the comparative example.

[0104] Example 1 1.21 28.36 1.02 96.23 Example 2 0.98 22.15 0.86 97.15 Example 3 1.56 35.82 1.35 94.86 Example 4 0.85 19.68 0.72 97.82 Comparative Example 1 1.25 29.12 1.05 96.18 Comparative Example 2 3.86 68.53 8.92 88.35 Comparative Example 3 2.73 45.26 1.13 92.67

[0105] As shown in Table 1, the matrices prepared in the four embodiments of the present invention all have excellent purification effects and structural integrity. The cell residue rate is less than 1.6% and the DNA residue is less than 36 ng / mg, which fully meets the industry standards for biomedical decellularized matrices. At the same time, the pore integrity rate is higher than 94%, which can completely preserve the natural three-dimensional porous structure of the lipid matrix and provide a good carrier environment for cell adhesion and proliferation.

[0106] Comparison of the data from each group reveals that Example 4 exhibits the best overall purification effect, with a cell residue rate of only 0.85%, DNA residue of 19.68 ng / mg, and lipid residue of 0.72 wt%. This is attributed to the use of the maximum material-to-liquid ratio, the longest decellularization reaction time, and the high-frequency reagent replacement process employed in this example, which effectively removes impurities such as cells, nucleic acids, and lipids from the matrix. Example 2, employing a carbodiimide crosslinking modification system, shows a slightly lower purification effect but maintains stable overall performance. Example 3, using the lower limit conditions of the process parameters, exhibits purification indicators that are slightly lower than those of the other examples, but still meet medical standards, demonstrating the good applicability of the process parameter range of this invention.

[0107] Comparative Example 1 omitted the low immunogenicity modification step, and its cell residue, DNA, and lipid residue indicators were basically the same as those in Example 1, indicating that the modification step only optimized the matrix immunogenicity and did not affect the matrix decellularization and delipidation purification effect. The two processes are independent of each other and do not interfere with each other.

[0108] Comparative Example 2, which omitted the defatting step, showed a significant deterioration in various purification indicators. The residual lipid content soared to 8.92 wt%, the residual DNA content exceeded medical standards by more than twice, the cell residue rate increased significantly, and the pore integrity rate dropped to 88.35%. This fully demonstrates that the defatting step is the core step in removing a large amount of lipid impurities from the adipose matrix and assisting in the removal of cell residues. Residual lipids can encapsulate cell debris and nucleic acid impurities, hindering the action of decellularization agents and damaging the matrix pore structure. It is an indispensable key process.

[0109] In Comparative Example 3, the decellularization process was carried out without changing the reagents. Compared with Example 2, the cell residue rate and DNA residue amount increased significantly, and the pore integrity rate decreased. This is because the decellularization reagents are consumed and become ineffective during the continuous reaction. In the later stage of the reaction, the reagent activity is insufficient and cannot completely lyse the residual cells and nucleic acid impurities, resulting in a decrease in purification effect. This proves that changing the reagents regularly during the decellularization process is a key auxiliary process to ensure the thoroughness of decellularization.

[0110] Table 2: Comparison of immunogenicity performance of the matrix in each example and the comparative example

[0111] Example 1 42.36 38.52 2.1 3.25 Example 2 35.18 32.16 1.8 2.68 Example 3 48.25 45.31 2.4 3.82 Example 4 30.62 28.75 1.5 2.15 Comparative Example 1 89.73 85.26 5.8 9.63 Comparative Example 2 72.35 68.92 4.6 7.28 Comparative Example 3 65.82 62.15 4.2 6.85

[0112] Immunogenicity is the core optimization indicator of this invention. The lower the secretion levels of inflammatory factors TNF-α and IL-6, the lower the immunogenicity score and the smaller the cell rejection rate, indicating better matrix biocompatibility and lower immunogenicity. As shown in Table 2, after specific low-immunogenicity modification, the secretion levels of inflammatory factors in the four embodiments of this invention were significantly reduced, the immunogenicity scores were all below 2.5, and the cell rejection rate was below 4%, exhibiting extremely low immune rejection risk and meeting the requirements of high-end biomedical materials.

[0113] The optimization effects of different modification systems varied significantly: Example 4 used a high-concentration composite alcohol-fatty alcohol polyoxyethylene ether modification system, which had the lowest secretion of matrix inflammatory factors and an immunogenicity score of only 1.5 after sufficient reaction, showing the best modification effect; the carbodiimide crosslinking modification system in Example 2 could effectively crosslink active antigen sites on the matrix surface and block immune recognition sites, showing the second best immune optimization effect; the glutaraldehyde crosslinking system in Example 3 had a mild process, was suitable for short-time reaction scenarios, and performed slightly better than conventional alcohol modification systems; the basic modification system in Example 1 had stable performance and could meet routine medical needs.

[0114] Comparative Example 1, which was not subjected to low-immunomodification, had TNF-α and IL-6 secretion levels more than twice that of Example 1, and its immunogenicity score increased significantly to 5.8 points, with a cell rejection rate of 9.63%. This directly proves that the S5 modification step of the present invention is the core key step in reducing matrix immunogenicity, which can effectively block trace antigen sites remaining in decellularized matrix, modify the physicochemical properties of matrix surface, and significantly reduce the body's immune rejection response.

[0115] Comparative Example 2, lacking defatting treatment, had a large amount of residual lipid impurities and cell antigens in its matrix. Even after modification, it still exhibited a high level of inflammatory response and immune rejection, with immune performance far inferior to that of the other examples. This demonstrates that the initial defatting and decellularization purification process is the foundation for low-immune modification. Only by thoroughly removing impurities can the modification effect be fully realized.

[0116] Comparative Example 3 involved decellularization without reagent replacement, resulting in trace amounts of cellular antigens and nucleic acid fragments remaining in the matrix. The amount of residual antigens was higher than in Example 2, leading to a deterioration in immunogenicity indicators. This demonstrates that the thoroughness of the initial purification process directly determines the final immunogenicity of the matrix, and that multi-step synergistic optimization is necessary to achieve ultra-low immunogenicity.

[0117] Table 3: Comparison of matrix stability and biocompatibility of different modified systems

[0118] Example 1 92.36 88.62 2.15 Level 1 (Excellent) Example 2 94.15 91.35 1.86 Level 1 (Excellent) Example 3 90.82 86.28 2.32 Level 1 (Excellent) Example 4 95.68 93.75 1.65 Level 1 (Excellent) Comparative Example 1 85.23 76.32 2.85 Level 2 (Good) Comparative Example 2 81.56 71.85 3.26 Level 2 (General) Comparative Example 3 87.32 79.63 2.58 Level 2 (Good)

[0119] As shown in Table 3, the matrix prepared in all embodiments of the present invention has a 7-day structure retention rate of over 90%, a cell adhesion rate of over 86%, a mild and controllable degradation rate, no rapid degradation or degradation lag issues, and biocompatibility reaches the first-class excellent level, which is suitable for the needs of human soft tissue repair.

[0120] Example 4, after high-parameter composite modification, exhibited the best matrix structural stability, with a 7-day structure retention rate of 95.68%, the highest cell adhesion rate, and the mildest degradation rate. This was attributed to the thorough modification reaction, which optimized the matrix surface microstructure, enhanced matrix fiber cross-linking, improved structural stability, and improved surface hydrophilicity, facilitating cell adhesion and colonization. Example 2, with its carbodiimide cross-linking modification, effectively strengthened the internal fiber cross-linking of the matrix, demonstrating excellent structural stability and cell compatibility. Examples 3 and 1 meet the requirements for conventional repair scenarios, exhibiting stable and reliable performance.

[0121] All comparative samples only met the second-level biocompatibility standard, and their structural stability and cell adhesion performance were significantly inferior to those of the examples. Comparative Example 1, with its unmodified matrix surface structure and low fiber cross-linking degree, was prone to structural damage after implantation, resulting in decreased cell adhesion, excessively rapid degradation, and inability to support tissue repair in the long term. Comparative Example 2 contained a large amount of residual lipids and impurities, with disordered matrix pore structure and reduced cell adhesion sites. At the same time, the impurities slightly inhibited cell growth, resulting in the worst structural stability. Comparative Example 3 was not completely decellularized, leaving trace amounts of antigenic impurities, which slightly affected cell adhesion and matrix structural stability, with performance between Comparative Example 1 and Comparative Example 2.

[0122] The data from the three sets of tables fully validate that this invention, through optimized processes including defatting, virus inactivation, decellularization, and low immunomodification, and the synergistic cooperation of multiple steps, can prepare a high-purity, highly stable, low-immunogenic, and highly biocompatible decellularized lipid matrix. The initial defatting and thorough decellularization processes remove matrix impurities, providing a pure substrate for the modification reaction. The specific low immunomodification system blocks antigen sites and optimizes the matrix structure and surface properties, ultimately achieving a significant reduction in matrix immunogenicity while fully preserving its natural three-dimensional structure and bioactivity. Compared to traditional unmodified, defatted, and incompletely decellularized matrix products, the overall performance is significantly improved, possessing extremely high medical application value.

[0123] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for modifying acellular adipose matrix with low immunogenicity, characterized in that, The method includes: S1: The raw adipose tissue is cleaned and crushed to obtain rinsed adipose tissue; S2: The rinsed adipose tissue is pretreated, the pretreatment including centrifugation, collection of the fat layer, homogenization and posttreatment, to obtain a defatted solid layer; S3: Perform virus inactivation treatment on the degreased solid layer, wherein the virus inactivation treatment includes mixing the degreased solid layer with a virus inactivation reagent to obtain a virus-inactivated solid layer. S4: Decellularize the solid layer after virus inactivation, wherein the decellularization process includes mixing the solid layer after virus inactivation with a decellularization reagent to obtain a decellularized lipid matrix; S5: The decellularized adipose matrix is ​​subjected to low immunogenicity modification treatment to obtain a low immunogenicity decellularized adipose matrix.

2. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S1, the cleaning and crushing of the adipose tissue raw material includes: After the adipose tissue material is allowed to stand to remove the lower layer of swelling fluid, it is rinsed with physiological saline 2-5 times, and then the rinsed adipose tissue material is mechanically crushed to obtain rinsed adipose tissue.

3. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S2, the centrifugation and collection of the fat layer includes: The rinsed adipose tissue was centrifuged at 8000-12000g for 1-5 minutes to obtain a mixture with a three-layer structure, wherein 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. Remove the upper oil layer and the lower water layer, and collect the intermediate layer.

4. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S2, the homogenization and post-processing include: The intermediate layer is mechanically homogenized at a speed of 8000-15000 r / min for 1-5 minutes to obtain a homogenized product; The homogenized product is centrifuged at 8000-12000g for 5-15 minutes to remove the upper oil layer and collect the lower solid layer.

5. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S2, obtaining the degreased solid layer further includes: The lower solid layer is mixed with a degreasing agent at a mass ratio of 1:5 to 1:20 and treated under shaking conditions for 2-8 hours. The degreasing agent is an aqueous solution containing 1-100 mmol / L of alkaline substance, 50-95% by volume of a first alcohol substance, and 0.1-5% by volume of polyethylene glycol ether. The treated mixture is centrifuged or allowed to stand before the liquid layer is removed and the solid layer is collected to obtain the degreased solid layer.

6. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S3, mixing the defatted solid layer with the virus inactivation reagent includes: The defatted solid layer is mixed with the virus inactivation reagent at a mass ratio of 1:5 to 1:20 and treated under shaking conditions for 1-6 hours. The virus inactivation reagent is an aqueous solution containing 0.1-2% peroxide and 2-10% second alcohol by volume. The treated mixture is centrifuged or allowed to stand before the liquid layer is removed and the solid layer is collected. The solid layer was washed sequentially with PBS solution and purified water by shaking to obtain the virus-inactivated solid layer.

7. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S4, mixing the virus-inactivated solid layer with the decellularization reagent includes: The solid layer after virus inactivation is mixed with the decellularization reagent at a mass ratio of 1:5 to 1:20 and treated under shaking conditions for 4-12 hours. The decellularization reagent is an aqueous solution containing 0.5-2% by volume of a surfactant, which is selected from one or more of SDS, Triton X-100 and sodium deoxycholate. During the process, the decellularization reagent should be replaced every 2-6 hours; The treated mixture is centrifuged or allowed to stand before the liquid layer is removed and the solid layer is collected. The solid layer was washed sequentially with PBS solution and purified water by shaking to obtain the decellularized lipid matrix.

8. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S5, the modifying agent is an aqueous solution containing 0.5-5% by volume fatty alcohol polyoxyethylene ether and 1-10% by volume a third alcohol. The decellularized lipid matrix and the modifying agent were mixed at a mass ratio of 1:5 to 1:15 and reacted with shaking at 25-37°C for 6-18 hours. The mixture after the reaction was centrifuged or allowed to stand before the liquid layer was removed and the solid layer was collected. The solid layer was then washed with PBS solution and purified water in sequence to obtain the low-immunogenic decellularized lipid matrix.

9. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S5, the modifying reagent is a MES buffer containing 0.1-2% by mass of N-hydroxysuccinimide and 0.1-1% by mass of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, wherein the concentration of the MES buffer is 10-100 mM and the pH is 5.0-6.

5. The decellularized lipid matrix and the modifying agent were mixed at a mass ratio of 1:8 to 1:20 and reacted with shaking at 20-30°C for 2-12 hours. The mixture after the reaction was centrifuged or allowed to stand before the liquid layer was removed and the solid layer was collected. The solid layer was then washed with PBS solution and purified water in sequence to obtain the low-immunogenic decellularized lipid matrix.

10. The method for modifying acellular adipose matrix with low immunogenicity according to claim 1, characterized in that, In step S5, the modifying reagent is a phosphate buffer containing 0.1-1% glutaraldehyde and 1-5% a fourth alcohol by volume, wherein the concentration of the phosphate buffer is 10-50 mM and the pH is 7.2-7.

8. The decellularized lipid matrix and the modifying agent were mixed at a mass ratio of 1:10 to 1:20 and reacted with shaking at 25-37°C for 1-8 hours. The mixture after the reaction was centrifuged or allowed to stand before the liquid layer was removed and the solid layer was collected. The solid layer was then washed with PBS solution and purified water in sequence to obtain the low-immunogenic decellularized lipid matrix.

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