A freeze-dried composition of adipose acellular matrix and its preparation method

Abstract

The present invention provides a freeze-dried composition of adipose acellular matrix. Specifically, the freeze-dried composition is prepared by freeze-drying a formulated adipose acellular matrix emulsion obtained by mixing the adipose acellular matrix with an auxiliary agent. The auxiliary agent includes auxiliary agent A and optionally auxiliary agent B. Auxiliary agent A includes one or more of mannitol, trehalose, and sucrose; auxiliary agent B includes one or more of glycerol, polyethylene glycol, polyvinyl alcohol, carbomer, sodium alginate, and arabic gum. The freeze-dried composition of adipose acellular matrix of the present invention solves the problem that it is difficult for the adipose acellular matrix to be injected through a small-diameter needle after freeze-drying. The particle size distribution after reconstitution is controllable, the stability of the solution system after reconstitution is good, and it has good biocompatibility and can promote the proliferation of adipocytes.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly relates to a freeze-dried composition of adipose acellular matrix and a preparation method thereof. Background Art

[0002] The adipose acellular matrix contains active components such as active proteins, growth factors, and polymorphic collagen. It not only has good biocompatibility but also has the effects of inducing the proliferation and differentiation of adipocytes. Injecting the adipose acellular matrix has good effects in treating soft tissue volume loss or skin quality improvement caused by various traumas, diseases, and aging.

[0003] Needles with different gauges are suitable for injecting different layers of soft tissues. For example, needles with a gauge of 20-25G can be used for deep injection into the subcutaneous tissue of the face; needles with a gauge of 25-30G can be used for injecting into the dermal tissue of the face; needles with a gauge of 30G and above can be used for injecting into the superficial skin. The freeze-drying process can retain the activity of the adipose acellular matrix to the greatest extent and extend its storage time. However, after freeze-drying treatment, the adipose acellular matrix will undergo tangling and aggregation, making it difficult to redissolve evenly and unable to be injected through small-bore needles. Since different-bore needles are respectively used for injecting different layers of soft tissues, the products after conventional freeze-drying treatment limit their clinical applications.

[0004] Therefore, it is necessary to provide a new freeze-dried composition of adipose acellular matrix and a preparation method thereof to reduce the adverse effects brought about during the freeze-drying process and solve the deficiencies of the prior art. Summary of the Invention

[0005] The purpose of the present invention is to provide a freeze-dried composition of adipose acellular matrix and a preparation method thereof.

[0006] In the first aspect of the present invention, a freeze-dried composition of adipose acellular matrix is provided. The freeze-dried composition is prepared by the following method, and the method includes the steps:

[0007] a. Mix the adipose acellular matrix with an auxiliary agent to obtain a formulated adipose acellular matrix emulsion;

[0008] b. Freeze-dry the formulated adipose acellular matrix emulsion to obtain the freeze-dried composition of adipose acellular matrix;

[0009] Wherein, the auxiliary agent includes auxiliary agent A and optionally auxiliary agent B; the auxiliary agent A is saccharides and polyols, preferably including one or more of mannitol, trehalose, and sucrose; the auxiliary agent B includes one or more of glycerol, polyethylene glycol, polyvinyl alcohol, carbomer, sodium alginate, and arabic gum.

[0010] In another preferred example, the auxiliary agent A is mannitol.

[0011] In another preferred example, the auxiliary agent B is selected from the group consisting of glycerol, polyethylene glycol, or a combination thereof.

[0012] In another preferred example, the molecular weight of the polyethylene glycol is 100 - 2000, preferably 100 - 1000, such as 200, 400, 600, 800.

[0013] In another preferred example, in the prepared acellular adipose matrix emulsion, the concentration of the acellular adipose matrix is 0.1 - 40 mg / mL, preferably 0.5 - 30 mg / mL, more preferably 1 - 25 mg / mL, such as 3 mg / mL, 5 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL.

[0014] In another preferred example, in the prepared acellular adipose matrix emulsion, the mass concentration of the auxiliary agent A is 0.1 - 30 mg / mL, preferably 0.5 - 25 mg / mL, more preferably 0.5 - 20 mg / mL, such as 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL.

[0015] In another preferred example, in the prepared acellular adipose matrix emulsion, the concentration of the auxiliary agent B is 10 - 500 μL / mL, preferably 20 - 200 μL / mL, more preferably 50 - 200 μL / mL, such as 100 μL / mL.

[0016] In another preferred example, when the prepared acellular adipose matrix emulsion contains the auxiliary agent B, the concentration of the acellular adipose matrix is 0.1 - 40 mg / mL, preferably 0.5 - 30 mg / mL, more preferably 1 - 25 mg / mL, such as 3 mg / mL, 5 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL.

[0017] In another preferred example, when the prepared acellular adipose matrix emulsion does not contain the auxiliary agent B, the concentration of the acellular adipose matrix is 0.1 - 30 mg / mL, preferably 0.5 - 25 mg / mL, more preferably 1 - 20 mg / mL, such as 3 mg / mL, 5 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL.

[0018] In another preferred example, the prepared acellular adipose matrix emulsion contains 1 - 5 mg / mL of acellular adipose matrix and 0.5 - 15 mg / mL of mannitol.

[0019] In another preferred example, the formulated adipose acellular matrix emulsion contains 1 - 5 mg / mL of adipose acellular matrix and 5 - 15 mg / mL of alginic acid.

[0020] In another preferred example, the formulated adipose acellular matrix emulsion contains 1 - 5 mg / mL of adipose acellular matrix, 5 - 15 mg / mL of mannitol, and 50 - 200 μL / mL of glycerol.

[0021] In another preferred example, the formulated adipose acellular matrix emulsion contains 1 - 40 mg / mL of adipose acellular matrix, 5 - 15 mg / mL of mannitol, and 50 - 200 μL / mL of PEG 400.

[0022] In another preferred example, the formulated adipose acellular matrix emulsion contains 1 - 20 mg / mL of adipose acellular matrix and 5 - 15 mg / mL of mannitol.

[0023] In another preferred example, the adipose acellular matrix is prepared by the following method, which includes:

[0024] S1: Cleaning

[0025] Provide a raw material of adipose tissue, crush the raw material of adipose tissue, and perform rinsing to obtain the rinsed adipose tissue;

[0026] S2: Pretreatment

[0027] (1) Centrifuge the rinsed adipose tissue to obtain a layered mixture, remove the upper oil layer and the lower water layer, and collect the middle layer;

[0028] (2) Homogenize the middle layer and perform post - treatment to obtain a solid layer;

[0029] (3) Rinse the solid layer in step (2) to obtain a solid layer;

[0030] Among them, the rinsing treatment includes mixing the solid layer in step (2) with a defatting reagent, and the defatting reagent is a mixed aqueous solution of an alkaline substance, a first alcohol substance, and a polyethylene glycol ether;

[0031] S3: Virus inactivation

[0032] Treat the solid layer in step S2(3) with a virus inactivation reagent to obtain a solid layer;

[0033] Among them, the virus inactivation treatment includes mixing the solid layer with a virus inactivation reagent, and the virus inactivation reagent includes an aqueous solution of a peroxide and a second alcohol substance;

[0034] S4: Decellularization

[0035] Treat the solid layer in step S3 with a decellularization reagent to obtain adipose decellularized matrix.

[0036] In another preferred example, no bioenzyme is added in the method.

[0037] In another preferred example, the total reaction time of the method is less than 72 hours, preferably less than 48 hours, more preferably less than 24 hours.

[0038] In another preferred example, step S1 further includes: allowing the adipose tissue raw material to stand still to remove the lower layer of swollen liquid.

[0039] In another preferred example, in step S1, rinsing is performed with physiological saline.

[0040] In another preferred example, in step S1, the number of rinsing times is 2 - 5 times, such as 3 times.

[0041] In another preferred example, step S1 includes: standing the adipose tissue upright and still, removing the swollen liquid, and then rinsing 3 - 5 times with physiological saline.

[0042] In another preferred example, in step (1), the rotation speed of the centrifugation is 200 - 20000g, preferably 2000 - 16000g, and most preferably 8000 - 12000g.

[0043] In another preferred example, in step (1), the centrifugation time is 1 - 15 min, preferably 1 - 10 min, more preferably 1 - 8 min, and most preferably 1 - 5 min.

[0044] In another preferred example, in step (1), the intermediate layer is an adipose layer containing adipose cells.

[0045] In another preferred example, in step (1), the layered mixture is divided into three layers, the upper layer is an oil layer, the intermediate layer is an adipose layer containing adipose cells, and the lower layer is a water layer.

[0046] In another preferred example, step (1) includes: centrifuging the rinsed adipose tissue at 10000g for 3 minutes to obtain a layered mixture, removing the upper oil layer and the lower water layer, and collecting the intermediate layer (i.e., the adipose layer containing adipose cells).

[0047] In another preferred example, in step (2), the homogenization is mechanical homogenization.

[0048] In another preferred example, in step (2), the homogenization treatment includes crushing and homogenizing through a homogenizer.

[0049] In another preferred example, the rotational speed of the homogenizer is 5000 - 15000 r / min, preferably 8000 - 15000 r / min, such as 10000 r / min, 12000 r / min.

[0050] In another preferred example, in the step (2), the time for the homogenization treatment is 0.5 - 15 min, preferably 1 - 10 min, more preferably 1 - 8 min, and most preferably 1 - 5 min.

[0051] In another preferred example, in the step (2), the rotational speed for centrifugation is 200 - 20000 g, preferably 2000 - 16000 g, and most preferably 8000 - 12000 g.

[0052] In another preferred example, in the step (2), the time for centrifugation is 2 - 20 min, preferably 5 - 15 min, and more preferably 10 min.

[0053] In another preferred example, in the step (2), the post - treatment includes centrifugal separation and the step of collecting the solid layer.

[0054] In another preferred example, the step (2) includes: homogenizing the intermediate layer obtained in step (1) at a rotational speed of 10000 r / min for 1 minute, then centrifuging at 10000 g and collecting the solid layer.

[0055] In another preferred example, in the step (3), the mixing includes oscillating mixing.

[0056] In another preferred example, in the step (3), the mass ratio of the solid layer in the step (2) to the degreasing reagent is 1:1 - 1:20, preferably 1:2 - 1:20, more preferably 1:5 - 1:20, such as 1:6, 1:8, 1:10.

[0057] In another preferred example, the time for the oscillating mixing is 0.5 - 8 h, preferably 2 - 8 h, such as 3 h, 4 h.

[0058] In another preferred example, the alkaline substance is selected from the group consisting of sodium hydroxide, sodium carbonate, and sodium bicarbonate, or a combination thereof.

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

[0060] In another preferred example, the polyethylene glycol ether is selected from the group consisting of fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene ether, sodium lauryl ether sulfate, and octylphenol polyoxyethylene ether, or a combination thereof.

[0061] In another preferred example, the concentration of the alkaline substance in the degreasing reagent is 1 mmol / L - 100 mmol / L.

[0062] In another preferred example, the volume fraction of the first alcohol substance in the degreasing reagent is 50 - 100%, preferably 50 - 95%.

[0063] In another preferred example, the volume fraction of the polyethylene glycol ether in the degreasing reagent is 0.1 - 5%.

[0064] In another preferred example, step (3) further includes: separating the mixture after the degreasing reagent, discarding the liquid layer, and obtaining the solid layer.

[0065] In another preferred example, the separation includes centrifugal separation, or directly sucking the liquid layer after standing, so as to discard the liquid layer.

[0066] In another preferred example, step (3) includes: adding a degreasing reagent to the solid layer in step (2) in a certain proportion, oscillating and mixing for a period of time, then discarding the liquid layer, and collecting the solid layer.

[0067] In another preferred example, step (3) includes: adding an aqueous solution of sodium hydroxide, isopropanol, and polyethylene glycol octyl phenyl ether to the solid layer in step (2) at a mass ratio of 1:10, oscillating and mixing for 3 hours, then discarding the liquid layer, and collecting the solid layer.

[0068] The preparation method of the acellular matrix needs to be selected according to the different characteristics of the target tissue used, and the process method is integrated according to the cell characteristics, density, and lipid content in the tissue. Since adipose tissue contains more than 80% lipids, the lipids adhere to the surface of the matrix, resulting in a decrease in the efficiency of the acellular agent treatment in the subsequent treatment process. The present invention can remove the vast majority of lipids in adipose tissue through a combined method of homogenization, centrifugation, and rinsing, greatly reducing the subsequent acellular treatment time and improving the preparation efficiency of the adipose acellular matrix.

[0069] In another preferred example, in step S3, the mixing includes oscillating and mixing.

[0070] In another preferred example, in step S3, the virus inactivation treatment includes adding the solid content to the virus inactivation reagent and mixing.

[0071] In another preferred example, the peroxide is selected from the group consisting of: hydrogen peroxide, peracetic acid, chlorine dioxide, ozone, or a combination thereof.

[0072] In another preferred example, the second alcohol substance is selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol, or a combination thereof.

[0073] In another preferred example, the volume fraction of the peroxide in the virus inactivating reagent is 0.05-3%, preferably 0.1-2%, more preferably 0.1-1%, such as 0.5%.

[0074] In another preferred example, the volume fraction of the alcohol in the virus inactivating reagent is 1-24%, preferably 2-20%, more preferably 2-10%, such as 4%, 5%, 8%.

[0075] In another preferred example, in the step S3, the mass ratio of the solid layer in the step S2(3) to the virus inactivating reagent is 1:1 to 1:20, preferably 1:2 to 1:20, more preferably 1:5 to 1:20, such as 1:8, 1:10.

[0076] In another preferred example, the time of the oscillating mixing is 0.5-6 h, preferably 1-6 h, such as 4 h.

[0077] In another preferred example, the step S3 further includes: separating the mixture after the virus inactivating reagent, discarding the liquid layer, and then washing the remaining solid layer to obtain a solid layer.

[0078] In another preferred example, the separation includes centrifugal separation, or optionally directly sucking the liquid layer after standing to discard the liquid layer.

[0079] In another preferred example, the washing includes: washing with PBS solution and / or water.

[0080] In another preferred example, the washing includes: first oscillating and washing with PBS solution, and then oscillating and washing with purified water.

[0081] In another preferred example, the step S3 includes: adding the solid layer in the step S2(3) into the virus inactivating reagent at a certain ratio for oscillating mixing, discarding the liquid layer after treatment, and successively oscillating and washing the remaining solid with PBS solution, purified water, and sodium chloride solution to obtain a solid layer.

[0082] In another preferred example, the step S3 includes: adding the solid layer in the step S2(3) into a peracetic acid / ethanol disinfectant with a concentration of 0.5% / 4% at a mass ratio of 1:10 for oscillating mixing for 4 hours, discarding the liquid layer after treatment, and successively oscillating and washing the remaining solid with PBS solution and purified water to obtain a solid layer.

[0083] In another preferred example, in the step S4, the decellularization treatment includes mixing the solid content with a decellularization reagent.

[0084] In another preferred example, the mixing includes oscillating mixing.

[0085] In another preferred example, in step S4, the decellularization treatment includes adding solid contents to a decellularization reagent and mixing them.

[0086] In another preferred example, in step S4, the decellularization treatment includes adding solid contents to a decellularization reagent, mixing for a period of time, replacing the decellularization reagent with a new one, and continuing to mix.

[0087] In another preferred example, the decellularization reagent is an aqueous solution of a surfactant.

[0088] In another preferred example, the surfactant is selected from one or more of non-ionic surfactants or ionic surfactants.

[0089] In another preferred example, the surfactant is selected from one or more of SDS, Triton X-100, and sodium deoxycholate.

[0090] In another preferred example, 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%.

[0091] In another preferred example, 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, 1:10.

[0092] In another preferred example, the time for oscillating and mixing is 1-12 h, preferably 4-12 h, for example 1 h, 2 h, 4 h, 8 h, 12 h.

[0093] In another preferred example, the mixing includes first subjecting to oscillating treatment for t1 hours, then changing the liquid, and then subjecting to oscillating treatment for t2 hours.

[0094] In another preferred example, t1 and t2 are each independently 2-6 h, for example 4 h.

[0095] In another preferred example, changing the liquid means replacing the decellularization reagent with a new one.

[0096] In another preferred example, changing the liquid includes: separating, discarding the liquid layer, and then adding a new decellularization reagent.

[0097] In another preferred example, step S4 further includes: separating the mixture after treatment with the decellularization reagent, discarding the liquid layer, and then washing the remaining solid layer to obtain a solid layer.

[0098] In another preferred example, the separation includes centrifugal separation, or optionally, directly aspirating the liquid layer after standing still, thereby discarding the liquid layer.

[0099] In another preferred example, the washing includes: washing with PBS solution and / or water.

[0100] In another preferred example, the washing includes: first oscillating and washing with PBS solution, and then oscillating and washing with purified water.

[0101] In another preferred example, step S4 includes: adding the solid layer from step S3 into a decellularization reagent at a certain ratio and oscillating and mixing, discarding the liquid layer after treatment, and successively oscillating and washing the remaining solid with PBS solution and then with purified water to obtain a solid layer.

[0102] In another preferred example, step S4 includes: adding the solid layer from step S3 into 1% TritonX-100 at a mass ratio of 1:10 and oscillating and mixing for 8 hours, changing the liquid once every 4 hours, discarding the liquid layer after treatment, and successively oscillating and washing the remaining solid with PBS solution and then with purified water to obtain a fat decellularized matrix.

[0103] In another preferred example, in the degreasing reagent treatment and virus inactivation reagent treatment, an optional post-treatment step of washing is further included.

[0104] In another preferred example, the washing includes successively washing with PBS solution and then with purified water.

[0105] In another preferred example, the fat decellularized matrix is an allogeneic or syngeneic fat decellularized matrix.

[0106] In another preferred example, the fat decellularized matrix does not contain cells and does not contain lipid droplets.

[0107] In another preferred example, the lipid droplet is an oil droplet released after the fat cells are broken.

[0108] In another preferred example, "not containing lipid droplets" means that in the fat decellularized biomaterial, the percentage of the volume of the oil droplets in the total liquid is less than 1%, preferably less than 0.5%, more preferably less than 0.1%.

[0109] In another preferred example, the cells are selected from the group consisting of: endothelial cells, adipose stem cells, macrophage blood cells, stromal cells.

[0110] In another preferred example, "not containing cells" means that the average number of cells in 1 mL of the fat decellularized biomaterial is ≤ 1, preferably ≤ 0.5, more preferably ≤ 0.1, or is 0.

[0111] In another preferred embodiment, the DNA content in the decellularized adipose matrix is reduced by more than 80%, preferably more than 90%, more preferably more than 95% compared to untreated adipocytes.

[0112] In another preferred embodiment, step a further comprises: physically disrupting the decellularized adipose matrix in water to obtain a decellularized adipose matrix emulsion.

[0113] In another preferred embodiment, the physical disruption comprises using one or more of a knife homogenizer, an emulsifier, an ultrasonic cell disruptor, a tissue grinder, a jet mill, and a high-pressure homogenizer for physical disruption.

[0114] In another preferred embodiment, the physical disruption comprises homogenization.

[0115] In another preferred embodiment, the homogenization comprises knife homogenization and high-pressure homogenization.

[0116] In another preferred embodiment, the homogenization comprises knife homogenization at 10,000 - 20,000 rpm for 3 - 10 min, followed by high-pressure homogenization at 500 - 2000 bar for 1 - 5 cycles.

[0117] In another preferred embodiment, the water is purified water.

[0118] In another preferred embodiment, the particle size of the decellularized adipose matrix emulsion is 0.5 - 1000 μm, preferably 10 - 500 μm.

[0119] In another preferred embodiment, in step b, freeze-drying comprises pre-freezing, sublimation drying, and desorption drying.

[0120] In another preferred embodiment, the pre-freezing comprises: first maintaining at 0 - -8 °C for 0.2 - 1 h, then at -15 - -30 °C for 2 - 5 h, and finally at -30 - -70 °C for 1 - 5 h.

[0121] In another preferred embodiment, the sublimation drying comprises: first maintaining at -30 - -70 °C for 10 - 25 h, then at 0 - -8 °C for 0.5 - 3 h.

[0122] In another preferred embodiment, the desorption drying comprises maintaining at 10 - 35 °C for 2 - 8 h.

[0123] In another preferred embodiment, in step b, it further comprises the step of injecting the prepared decellularized adipose matrix emulsion into a mold, followed by freeze-drying.

[0124] In another preferred embodiment, the mold is selected from the group consisting of trays of different specifications, vials, glass syringes, or combinations thereof.

[0125] In another preferred example, the lyophilized composition is prepared by the following method, which includes the steps:

[0126] a1. Homogenously mix the acellular adipose matrix with water to obtain an acellular adipose matrix emulsion;

[0127] a2. Mix the acellular adipose matrix emulsion with an auxiliary agent to obtain a formulated acellular adipose matrix emulsion;

[0128] b. Lyophilize the formulated acellular adipose matrix emulsion to obtain the acellular adipose matrix lyophilized composition.

[0129] In another preferred example, the acellular adipose matrix lyophilized composition does not contain cells and does not contain lipid droplets.

[0130] In another preferred example, the particle size D(0.1) of the acellular adipose matrix lyophilized composition is less than 26 μm, preferably 10 - 26 μm, more preferably 10 - 25 μm.

[0131] In another preferred example, the particle size D(0.5) of the acellular adipose matrix lyophilized composition is less than 135 μm, preferably 50 - 130 μm, more preferably 70 - 125 μm.

[0132] In another preferred example, the particle size D(0.9) of the acellular adipose matrix lyophilized composition is less than 400 μm, preferably 100 - 350 μm, more preferably 150 - 300 μm.

[0133] In another preferred example, the acellular adipose matrix lyophilized composition has one or more characteristics selected from the following groups:

[0134] (1) The reconstitution time of the acellular adipose matrix lyophilized composition is less than 30 min, preferably 5 - 25 min, more preferably 5 - 20 min;

[0135] (2) The stability time after reconstitution of the acellular adipose matrix lyophilized composition is greater than 10 min, preferably greater than 20 min, more preferably greater than 30 min;

[0136] (3) The acellular adipose matrix lyophilized composition can be injected through a 27G needle, preferably can be injected through a 30G needle, more preferably can be injected through a 32G needle;

[0137] (4) The cell proliferation effect of the acellular adipose matrix lyophilized composition has no obvious difference compared with that of the directly lyophilized composition, and preferably the cell proliferation effect is increased by 10%.

[0138] In the second aspect of the present invention, there is provided a method for preparing the freeze-dried composition of adipose acellular matrix as described in the first aspect of the present invention, the method comprising the steps of:

[0139] a. Mixing the adipose acellular matrix with an adjuvant to obtain a formulated adipose acellular matrix emulsion;

[0140] b. Freeze-drying the formulated adipose acellular matrix emulsion to obtain the freeze-dried composition of adipose acellular matrix;

[0141] Wherein, the adjuvant includes adjuvant A and optionally adjuvant B; the adjuvant A is saccharides and polyols, preferably including one or more of mannitol, trehalose, and sucrose; the adjuvant B includes one or more of glycerol, polyethylene glycol, polyvinyl alcohol, carbomer, sodium alginate, and arabic gum.

[0142] In the third aspect of the present invention, there is provided a use of the freeze-dried composition of adipose acellular matrix as described in the first aspect of the present invention for preparing a medical material or a medical aesthetic material, the medical material being used for (1) soft tissue repair and / or (2) promoting cell proliferation, and the medical aesthetic material being used for plastic filling.

[0143] In another preferred example, the soft tissue repair includes promoting the proliferation of adipocytes.

[0144] In another preferred example, the plastic filling includes promoting the proliferation of adipocytes.

[0145] In the fourth aspect of the present invention, there is provided a freeze-dried powder, comprising the freeze-dried composition of adipose acellular matrix as described in the first aspect of the present invention.

[0146] In another preferred example, the freeze-dried powder further includes a pharmaceutically acceptable carrier, such as a freeze-drying stabilizer, a freeze-drying protectant, etc.

[0147] In another preferred example, the freeze-dried powder can be dissolved into a freeze-dried injection after adding a pharmaceutically acceptable buffer solution.

[0148] In another preferred example, the pharmaceutically acceptable buffer solution includes one or more of normal saline, PBS, and water for injection.

[0149] 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 specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0150] Figure 1Shows the cross-sectional SEM images of the fat acellular matrix freeze-dried product in Comparative Example 2 under ×100, ×300, ×1k, and ×5k magnifications.

[0151] Figure 2 Shows the physical image of the fat acellular matrix freeze-dried composition added with 0.5% mannitol (5 mg / mL) in Example 3.

[0152] Figure 3 Shows the SEM images of the fat acellular matrix added with 0.5% mannitol (5 mg / mL) in Example 3 under ×100, ×300, ×1k, and ×5k magnifications.

[0153] Figure 4a Shows the microscopic observation image under 40× magnification before freeze-drying in Comparative Example 1.

[0154] Figure 4b Shows the microscopic observation image under 40× magnification after reconstitution in Comparative Example 2.

[0155] Figure 4c Shows the microscopic observation image under 40× magnification after reconstitution of the fat acellular matrix freeze-dried composition in Example 4. Detailed implementation mode

[0156] Through extensive and in-depth research, the present inventors have first discovered a fat acellular matrix freeze-dried composition, which solves the problem that the fat acellular matrix is difficult to inject through a small-diameter needle after freeze-drying. The particle size distribution of the fat acellular matrix freeze-dried composition of the present invention after reconstitution is controllable, and the stability of the solution system after reconstitution is good. It can pass through a very small-diameter needle, so it can be applied to the injection of superficial skin, and has good biocompatibility, and can promote the proliferation of fat cells. On this basis, the present invention is completed.

[0157] Fat acellular matrix freeze-dried composition

[0158] The present invention provides a fat acellular matrix freeze-dried composition.

[0159] The described fat acellular matrix freeze-dried composition has excellent biological properties and can promote the proliferation of fat cells.

[0160] The described fat acellular matrix freeze-dried composition can be dispersed into a particulate state after reconstitution within a certain time, preferably within 30 minutes to 5 minutes.

[0161] The cumulative 90% particle size D(0.9) of the particle size distribution of the described fat acellular matrix freeze-dried composition after reconstitution does not exceed 150% of the particle size before freeze-drying.

[0162] For the lyophilized composition of acellular adipose matrix, the particle size D(0.5) of 50% of the cumulative particle size distribution after reconstitution does not exceed 200% of the particle size before lyophilization.

[0163] For the lyophilized composition of acellular adipose matrix, after short-term reconstitution, the time for maintaining the particle state without sedimentation is 10 minutes to 60 minutes or more.

[0164] The lyophilized composition of acellular adipose matrix is composed of acellular adipose matrix particles, auxiliary agent A and auxiliary agent B.

[0165] The acellular adipose matrix particles include irregular shapes such as fragmentary, fibrous, and bamboo joint-like shapes.

[0166] The source of the acellular adipose matrix is one or more of human or mammalian adipose tissue and skin tissue.

[0167] The auxiliary agent A is saccharides and polyols, preferably one or several of mannitol, trehalose, and sucrose.

[0168] During the freeze-drying process, the auxiliary agent A is deposited on the surface of the acellular adipose matrix particles, reducing the mutual entanglement between fibrous particles; at the same time, it is rich in hydroxyl groups and can form hydrogen bonds with the acellular adipose matrix, thereby reducing the interaction between matrix particles.

[0169] The auxiliary agent B includes one or several of glycerol, polyethylene glycol, polyvinyl alcohol, carbomer, sodium alginate, and gum arabic.

[0170] The auxiliary agent B is usually a water-soluble small molecule or polymer. During the reconstitution process of the lyophilized composition, it provides surface tension and viscosity, enabling the lyophilized composition to be uniformly dispersed after reconstitution and maintaining the dispersed state to reduce sedimentation.

[0171] The combination of auxiliary agent A and auxiliary agent B can also play a good shaping role during the freeze-drying process, making the lyophilized composition have sufficient skeleton support strength, a plump appearance, and good porosity.

[0172] Preparation method of the lyophilized composition of acellular adipose matrix

[0173] The present invention provides a preparation method of a lyophilized composition of acellular adipose matrix, comprising the following steps:

[0174] S1. Prepare acellular adipose matrix microparticles;

[0175] S2. Prepare a mixed solution of acellular adipose matrix, auxiliary agent A, and optionally auxiliary agent B;

[0176] S3. Inject into a mold and obtain the lyophilized composition of acellular adipose matrix after freeze-drying.

[0177] Further, the method for decellularizing adipose tissue in step S1 includes, but is not limited to, processing steps such as washing, pretreatment, virus inactivation, decellularization, degreasing, etc.

[0178] Further, the method for preparing adipose-derived decellularized matrix microparticles in step S1 is physical fragmentation, including using a knife homogenizer, emulsifier, ultrasonic cell disruptor, tissue grinder, air jet mill, high-pressure homogenizer, etc.

[0179] Further, the particle size of the adipose-derived decellularized matrix microparticles in step S1 is 0.5 - 1000 μm, preferably 10 - 500 μm.

[0180] Further, the content of additive A in the mixed solution in step S2 is 0.01 - 20% (mass / volume fraction), preferably 0.01 - 1.5%.

[0181] Further, the content of additive B in the mixed solution in step S2 is 0.01 - 10% (mass / volume fraction), preferably 0.01 - 1.0%.

[0182] Further, the molds in step S3 include trays, vials, glass syringes of different specifications, etc.

[0183] Further, the freeze-drying step in step S3 includes three stages: pre-freezing, sublimation drying, and desorption drying.

[0184] Further, the adipose-derived decellularized matrix freeze-dried composition can be reconstituted with physiological saline, PBS, water for injection, etc. before clinical use.

[0185] Medical materials and medical aesthetic materials

[0186] The present invention provides a material containing an adipose-derived decellularized matrix freeze-dried composition. The said material includes (but is not limited to): medical materials, medical aesthetic materials, etc.

[0187] Typically, the said material is a medical material, and the said medical material includes the adipose-derived decellularized matrix freeze-dried composition as described in the present invention; and a pharmaceutically acceptable carrier.

[0188] In the present invention, the dosage forms of the medical materials include (but are not limited to) freeze-dried powder agents, injections.

[0189] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, or liquid fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" means that the components in the pharmaceutical composition and the active ingredient of the drug are admixed with each other without significantly reducing the drug efficacy.

[0190] It should be understood that in the present invention, there is no particular limitation on the carrier, and commonly used materials in the art can be selected, or prepared by conventional methods, or obtained commercially. 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 dodecyl sulfate), buffers, chelating agents, thickening agents, pH regulators, transdermal penetration enhancers, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, bacteriostatic agents, pyrogen-free water, etc.

[0191] Typically, in addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers. For example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers, and suspending agents.

[0192] The dosage form of the material should match the mode of administration. The materials of the present invention can also be used together with other co-therapeutic agents (including before, during, or after use). When using the materials of the present invention, a safe and effective amount of the drug is administered to the desired subject (such as a human or non-human mammal). The safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dose should also consider factors such as the route of administration and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0193] The main advantages of the present invention include:

[0194] The particle size distribution of the reconstituted fat acellular matrix freeze-dried composition of the present invention is controllable, the stability of the reconstituted solution system is good, and it can pass through a very small-diameter needle, so it can be suitable for injection into the superficial skin, and has good biocompatibility and can promote the proliferation of fat cells.

[0195] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0196] Testing and Methods

[0197] The reconstitution performance was tested, that is, the reconstitution performance was represented by its reconstitution time, the stable time after reconstitution, and the needle gauge that could pass through.

[0198] Reconstitution time: Reconstitute with 1 mL of normal saline, slowly inject along the wall until the lyophilized composition is completely covered. After the lyophilized composition is completely infiltrated, record the standing time, shake well with a wrist oscillation, and consider it completely reconstituted when there are no large agglomerates and the particle size is uniform to the naked eye.

[0199] Stable time after reconstitution: During injection, it is necessary to maintain the reconstituted matrix solution in a uniformly dispersed state. Use a syringe to aspirate 1 mL of the reconstituted matrix solution, and the time required for obvious sedimentation to occur after standing is the stable time after reconstitution.

[0200] Injection after reconstitution: The larger the needle gauge, the smaller its inner diameter, and the corresponding inner diameters are shown in Table 1:

[0201] Needles of different gauges are suitable for injection into different layers of soft tissues. For example, needles of 20-25G can be used for deep injection into the subcutaneous tissue of the face; needles of 25-30G can be used for injection into the dermal tissue of the face; needles of 30G and above can be used for injection into the superficial skin.

[0202] Table 1 Needle Gauge and Inner Diameter Size

[0203] Model 20G 22G 23G 25G 27G 30G 32G Inner diameter 0.60mm 0.41mm 0.34mm 0.26mm 0.21mm 0.16mm 0.11mm

[0204] Example 1. Preparation of Acellular Fat Matrix

[0205] 1. Stand the obtained fat upright and let it stand still. After removing the swelling fluid, rinse it 3 times with normal saline.

[0206] 2. Take the rinsed adipose tissue, place it in a centrifuge tube, and centrifuge it at 10000g for 3 minutes in a centrifuge to obtain a layered mixture. For the layered mixture, remove the upper oil layer and the lower water layer, and collect the middle layer (i.e., the fat layer containing adipocytes).

[0207] Homogenize the middle layer at a speed of 10000 r / min for 1 minute, and centrifuge at 10000g to collect the solid layer.

[0208] Add an aqueous solution of sodium hydroxide, isopropyl alcohol, and polyethylene glycol octyl phenyl ether to the solid layer at a mass ratio of 1:10, shake for 3 hours, discard the liquid layer, and collect the solid layer.

[0209] 3. Add the solid content obtained in step 2 to a peracetic acid / ethanol disinfectant with a concentration of 0.5% / 4% at a mass ratio of 1:10, shake for 4 hours, discard the liquid layer after treatment, and wash the solid content successively by shaking with PBS solution, purified water, and sodium chloride aqueous solution to obtain the solid content.

[0210] 4. Add the solid content obtained in step 3 to 1% TritonX-100 at a mass ratio of 1:10, shake for 8 hours, change the liquid once every 4 hours, discard the liquid layer after treatment, and wash the solid content successively by shaking with PBS solution and purified water to obtain the acellular adipose matrix.

[0211] Comparative Example 1:

[0212] Take 1 part of the washed acellular adipose matrix, add 20 parts of purified water, homogenize with a knife at 15000 rpm for 6 min, and then homogenize at a high pressure of 1000 bar for 3 cycles to prepare a matrix emulsion with a concentration of 3 mg / mL.

[0213] The matrix emulsion obtained in Comparative Example 1 has a stability time of 20 min and can be injected through a 32G needle.

[0214] Comparative Example 2:

[0215] Take the matrix emulsion prepared in Comparative Example 1, without adding any additives A and B, inject it into a mold, and perform vacuum freeze-drying according to the following freeze-drying parameters:

[0216] ① Pre-freezing: First, keep at -4°C for 0.5 h, then at -20°C for 3 h, and finally at -50°C for 2 h;

[0217] ② Sublimation drying: Keep at -45°C for 15 h, then at -5°C for 1 h;

[0218] ③ Desorption drying: Keep at 25°C for 5 h.

[0219] The freeze-dried product of the acellular adipose matrix obtained in Comparative Example 2 has a reconstitution time of 30 min, a stability time of 3 min after reconstitution, and can be injected through a 25G needle.

[0220] The SEM photograph of the freeze-dried product of the acellular adipose matrix obtained in Comparative Example 2 is as Figure 1 shown. It is observed that the sample is an uneven porous network structure, including fibrous and flaky structures. Due to factors such as the entanglement of fibers in the acellular adipose matrix particles after freeze-drying, it is difficult for the particles to return to the state before freeze-drying.

[0221] Example 2:

[0222] Take 1 part of the washed acellular adipose matrix, add 20 parts of purified water, and homogenize with a blade at 15,000 rpm for 6 min, then homogenize under high pressure at 1000 bar for 3 cycles to obtain a matrix emulsion.

[0223] Prepare an emulsion with an acellular adipose matrix content of 3 mg / mL and a mannitol content of 1 mg / mL using purified water to obtain a formulated acellular adipose matrix emulsion. Inject it into a mold and perform vacuum freeze-drying according to the following freeze-drying parameters:

[0224] ① Pre-freezing: First, keep at -4°C for 0.5 h, then at -20°C for 3 h, and finally at -50°C for 2 h;

[0225] ② Sublimation drying: First, keep at -45°C for 15 h, then at -5°C for 1 h;

[0226] ③ Desorption drying: Keep at 25°C for 5 h.

[0227] The freeze-dried composition of the acellular adipose matrix obtained in Example 1 has a reconstitution time of 30 min and a stability time after reconstitution of 10 min, and can be injected through a 27G needle.

[0228] Example 3:

[0229] When formulating the acellular adipose matrix emulsion, the mannitol content is 5 mg / mL, and other parameters are the same as in Example 2.

[0230] The freeze-dried composition of the acellular adipose matrix obtained in Example 3 has a reconstitution time of 20 min and a stability time after reconstitution of 20 min, and can be injected through a 27G needle. The reconstitution effect is better than that of Example 2.

[0231] The physical picture of the freeze-dried composition of the acellular adipose matrix obtained in Example 3 is as shown in Figure 2 shown, it is loose and porous, and it is not easy to collapse during the freeze-drying process. Its SEM photos under different magnifications are as shown in Figure 3 shown, and the sample is observed to be a uniform porous network structure.

[0232] Example 4:

[0233] When formulating the acellular adipose matrix emulsion, the mannitol content is 10 mg / mL, and other parameters are the same as in Example 2.

[0234] The freeze-dried composition of the acellular adipose matrix obtained in Example 4 has a reconstitution time of 10 min and a stability time after reconstitution of 30 min, and can be injected through a 32G needle. The reconstitution effect is better.

[0235] The comparative example 1 (lyophilized adipose acellular matrix emulsion before lyophilization), comparative example 2 (lyophilized product without additives), and example 4 (lyophilized composition with 1% mannitol added) were observed microscopically under a 40× magnification, as shown in Figure 4a , 4b and 4c respectively. It was observed that the matrix emulsion before lyophilization was in a uniformly dispersed granular state. After reconstitution, the matrix particles of the adipose acellular matrix lyophilized product without additives showed a more aggregated state, while the lyophilized composition with 1% mannitol added was more uniformly dispersed after reconstitution, with a better effect.

[0236] Example 5:

[0237] When preparing the adipose acellular matrix emulsion, the content of trehalose was 10 mg / mL, and other parameters were the same as those in Example 2.

[0238] The adipose acellular matrix lyophilized composition obtained in Example 5 had a reconstitution time of 20 min and a stability time after reconstitution of 30 min, and could be injected through a 32G needle.

[0239] Example 6:

[0240] When preparing the adipose acellular matrix emulsion, the content of glycine was 10 mg / mL, and other parameters were the same as those in Example 2.

[0241] The adipose acellular matrix lyophilized composition obtained in Example 6 had a reconstitution time of 40 min and a stability time after reconstitution of 5 min, and could be injected through a 25G needle.

[0242] Example 7:

[0243] When preparing the adipose acellular matrix emulsion, the content of gelatin was 10 mg / mL, and other parameters were the same as those in Example 2.

[0244] The adipose acellular matrix lyophilized composition obtained in Example 7 had a reconstitution time of 40 min and a stability time after reconstitution of 5 min, and could be injected through a 25G needle.

[0245] Example 8:

[0246] When preparing the concentration of the adipose acellular matrix emulsion, the content of mannitol was 10 mg / mL and the content of glycerol was 10 μL / mL, and other parameters were the same as those in Example 2.

[0247] The adipose acellular matrix lyophilized composition obtained in Example 8 had a reconstitution time of 5 min and a stability time after reconstitution of 50 min, and could be injected through a 32G needle.

[0248] Example 9:

[0249] When formulating the concentration of the acellular adipose matrix emulsion, the mannitol content is 10 mg / mL, the polyethylene glycol (PEG 400) content is 10 μL / mL, and other parameters are the same as those in Example 2.

[0250] The acellular adipose matrix freeze-dried composition obtained in Example 9 has a reconstitution time of 5 min, a stable time after reconstitution of 60 min, and can be injected through a 32G needle.

[0251] Example 10:

[0252] When formulating the acellular adipose matrix emulsion, the concentration of the acellular adipose matrix is adjusted to 12 mg / mL, the mannitol content is 10 mg / mL, and other parameters are the same as those in Example 2.

[0253] The acellular adipose matrix freeze-dried composition obtained in Example 10 has a reconstitution time of 20 min, a stable time after reconstitution of 30 min, and can be injected through a 30G needle.

[0254] Example 11:

[0255] When formulating the acellular adipose matrix emulsion, the concentration of the acellular adipose matrix is adjusted to 36 mg / mL, the mannitol content is 10 mg / mL, and other parameters are the same as those in Example 2.

[0256] The acellular adipose matrix freeze-dried composition obtained in Example 11 has a reconstitution time of 30 min. Due to the too high matrix concentration, it cannot be completely mixed and shows many lumps.

[0257] Example 12:

[0258] When formulating the acellular adipose matrix emulsion, the concentration of the acellular adipose matrix is adjusted to 12 mg / mL, the mannitol content is 10 mg / mL, the polyethylene glycol (PEG 400) content is 10 μL / mL, and other parameters are the same as those in Example 2.

[0259] The acellular adipose matrix freeze-dried composition obtained in Example 12 has a reconstitution time of 10 min, a stable time after reconstitution of 30 min, and can be injected through a 30G needle.

[0260] Compared with Example 9, after adding adjuvant B (1% PEG400), the reconstitution situation is improved.

[0261] Example 13:

[0262] When formulating the acellular adipose matrix emulsion, the concentration of the acellular adipose matrix is adjusted to 36 mg / mL, the mannitol content is 10 mg / mL, the polyethylene glycol (PEG 400) content is 10 μL / mL, and other parameters are the same as those in Example 1.

[0263] The acellular adipose matrix freeze-dried composition obtained in Example 12 has a reconstitution time of 20 min and a stability time after reconstitution of 30 min, and can be injected through a 27G needle.

[0264] The particle size distributions of some of the examples were tested after reconstitution, and the results are shown in Table 2.

[0265] Table 2 Particle size distribution of the acellular adipose matrix freeze-dried composition after reconstitution

[0266]

[0267] Comparative Example 1 is the matrix emulsion before freeze-drying, and the particle size of Comparative Example 2 dispersed after freeze-drying without adding any additives is much larger than that of Comparative Example 1, so the effect of passing through the needle after reconstitution is poor.

[0268] Example 3 is an acellular adipose matrix freeze-dried composition added with mannitol at a concentration of 10 mg / mL. The dispersed particle size is slightly larger than that of Comparative Example 1 and is in the range of 100-150%, so the effect of passing through the needle after reconstitution is better.

[0269] Examples 5 and 6 are added with glycine and gelatin at a concentration of 10 mg / mL respectively. Their maximum particle size D(1) is in the range of 632.456-709.627 μm, and the volume average particle size is more than twice that before freeze-drying, so the reconstitution effect is poor.

[0270] Example 8 is an acellular adipose matrix freeze-dried composition added with additive A (10 mg / mL mannitol) and additive B (10 μL / mL PEG400) at the same time. Its dispersed particle size is also relatively close to that of Comparative Example 1 before freeze-drying.

[0271] Examples 9 and 10 are both added with 10 mg / mL mannitol, but their matrix concentrations are relatively high, 12 mg / mL and 36 mg / mL respectively, resulting in a relatively high dispersed particle size, especially the particle size of Example 10 is high and the dispersion effect is poor.

[0272] The matrix concentration of Example 12 is 36 mg / mL, and additive A (10 mg / mL mannitol) and additive B (10 μL / mL PEG400) are added at the same time. Compared with Example 10, the dispersion effect is significantly improved.

[0273] According to the above conditions, the reconstitution of the acellular adipose matrix freeze-dried composition is shown in Table 3.

[0274] Table 3 Reconstitution of the acellular adipose matrix freeze-dried composition under various conditions

[0275]

[0276] According to the above conditions, several of them were selected for cell proliferation assays. The cells used were adipocytes, and the detection method was CCK-8. The experimental results showed that the acellular adipose tissue matrix freeze-dried composition had a good effect on promoting cell proliferation. The results are shown in Table 4.

[0277] Table 4 Results of cell proliferation assays of acellular adipose tissue matrix freeze-dried composition under various conditions

[0278]

[0279] It can be seen that after the addition of the auxiliary agent, compared with the freeze-dried composition without the auxiliary agent, the performance of promoting cell proliferation was not weakened, and even increased to a certain extent.

[0280] In summary, the present invention provides an acellular adipose tissue matrix freeze-dried composition, which solves the problem that it is difficult for the acellular adipose tissue matrix to be injected through a small-caliber needle after freeze-drying. The particle size distribution of the acellular adipose tissue matrix freeze-dried composition of the present invention after reconstitution is controllable, and the stability of the reconstituted solution system is good. Moreover, it has good biocompatibility and can promote adipocyte proliferation.

Claims

1. A freeze-dried fat decellularized matrix composition, characterized in that: The freeze-dried composition is prepared by the following method, which comprises the steps of: a. mixing the adipose decellularized matrix with an auxiliary agent to obtain a formulated adipose decellularized matrix emulsion; b. freeze-drying the prepared adipose decellularized matrix emulsion to obtain the adipose decellularized matrix freeze-dried composition; Wherein, the adipose decellularized matrix is ​​prepared by the following method, which comprises: S1: Cleaning Providing a fat tissue raw material, breaking the fat tissue raw material, and rinsing it to obtain rinsed fat tissue; S2: Pre-treatment (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; (2) homogenizing the intermediate layer and performing post-processing to obtain a solid layer; (3) rinsing the solid layer in step (2) to obtain a solid layer; 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; S3: Virus inactivation Treating the solid layer in step (3) of step S2 with a virus inactivating agent to obtain a solid layer; Wherein, the virus inactivation treatment comprises mixing the solid layer with a virus inactivation agent, wherein the virus inactivation agent comprises an aqueous solution of peroxide, chlorine dioxide or ozone and a second alcohol substance; S4: Decellularization The solid layer in step S3 is treated with a decellularizing agent, the mixture treated with the decellularizing agent is separated, the liquid layer is discarded, and the remaining solid layer is washed to obtain a solid layer, i.e., adipose decellularized matrix; Wherein, the alkaline substance is selected from the group consisting of sodium hydroxide, sodium carbonate and sodium bicarbonate, or a combination thereof; The first alcohol substance is selected from the group consisting of 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; The peroxide is selected from the group consisting of hydrogen peroxide, peracetic acid, or a combination thereof; The second alcohol substance is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, or a combination thereof; The decellularization reagent is an aqueous solution of a surfactant, and the surfactant is selected from one or more of SDS, TritonX-100, and sodium deoxycholate; The auxiliary agent includes auxiliary agent A and optionally auxiliary agent B; The auxiliary agent A is one or more of mannitol and trehalose; The auxiliary agent B is one or more of glycerol and polyethylene glycol; When the formulated adipose decellularized matrix emulsion does not contain additive B, the concentration of the adipose decellularized matrix is ​​0.1-15 mg / mL; When the formulated adipose decellularized matrix emulsion contains auxiliary agent B, the concentration of the adipose decellularized matrix is ​​0.1-40 mg / mL.

2. The adipose decellularized matrix freeze-dried composition according to claim 1, characterized in that: The auxiliary agent A is mannitol.

3. The adipose decellularized matrix freeze-dried composition according to claim 1, characterized in that: The alkaline substance is sodium hydroxide.

4. The freeze-dried fat decellularized matrix composition according to claim 1, characterized in that: The formulated adipose decellularized matrix emulsion contains auxiliary agent B, and the concentration of the adipose decellularized matrix is ​​3-12 mg / mL.

5. The adipose decellularized matrix freeze-dried composition according to claim 1, characterized in that: The formulated adipose decellularized matrix emulsion contains auxiliary agent B, and the concentration of the adipose decellularized matrix is ​​20-30 mg / mL.

6. The adipose decellularized matrix freeze-dried composition according to claim 1, characterized in that: The first alcohol substance is isopropanol.

7. The adipose decellularized matrix freeze-dried composition according to claim 1, characterized in that: In the formulated adipose decellularized matrix emulsion, the mass concentration of the auxiliary agent A is 5-20 mg / mL.

8. The freeze-dried fat decellularized matrix composition according to claim 7, characterized in that: In the formulated adipose decellularized matrix emulsion, the mass concentration of the auxiliary agent A is 10-15 mg / mL.

9. The adipose decellularized matrix freeze-dried composition according to claim 1, characterized in that: The polyethylene glycol ether is polyethylene glycol octylphenyl ether.

10. The adipose decellularized matrix freeze-dried composition according to claim 1, characterized in that: In the formulated adipose decellularized matrix emulsion, the concentration of the auxiliary agent B is 10-100 μL / mL.

11. The freeze-dried adipose decellularized matrix composition according to claim 10, characterized in that: In the formulated adipose decellularized matrix emulsion, the concentration of the auxiliary agent B is 10-50 μL / mL.

12. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The peroxide is peracetic acid.

13. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The formulated adipose decellularized matrix emulsion contains 1-5 mg / mL adipose decellularized matrix and 0.5-15 mg / mL mannitol; Alternatively, the formulated adipose decellularized matrix emulsion contains 1-5 mg / mL adipose decellularized matrix and 5-15 mg / mL alginate; Alternatively, the formulated adipose decellularized matrix emulsion comprises 1-5 mg / mL adipose decellularized matrix, 5-15 mg / mL mannitol and 50-200 μL / mL glycerol; Alternatively, the formulated adipose decellularized matrix emulsion comprises 1-40 mg / mL adipose decellularized matrix, 5-15 mg / mL mannitol and 50-200 μL / mL PEG 400; Alternatively, the formulated adipose decellularized matrix emulsion contains 1-20 mg / mL adipose decellularized matrix and 5-15 mg / mL mannitol.

14. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The step (2) comprises: homogenizing the intermediate layer obtained in step (1) at a speed of 10,000 r / min for 1 minute, then centrifuging at 10,000 g to collect the solid layer.

15. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The adipose decellularized matrix contains no cells and no lipid droplets; The “free of fat droplets” means that in the adipose decellularized matrix, the volume of oil droplets accounts for less than 1% of the total liquid; the “free of cells” means that the average number of cells in 1 mL of adipose decellularized matrix is ​​≤1.

16. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The method has one or more features selected from the following group: The second alcohol substance is ethanol; The decellularization reagent is TritonX-100.

17. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The step a further comprises: physically crushing the adipose decellularized matrix in water to obtain adipose decellularized matrix emulsion, The physical disruption includes physical disruption using one or more of a knife homogenizer, an emulsifier, an ultrasonic cell disruptor, a tissue grinder, a jet mill, and a high-pressure homogenizer.

18. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: In the step b, freeze drying includes pre-freezing, sublimation drying and analytical drying.

19. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The adipose decellularized matrix freeze-dried composition has one or more characteristics selected from the following group: (1) The reconstitution time of the adipose decellularized matrix freeze-dried composition is less than 30 min; (2) The stability time of the adipose decellularized matrix freeze-dried composition after reconstitution is greater than 10 minutes; (3) The adipose decellularized matrix freeze-dried composition can be injected through a 27G needle; (4) There is no significant difference in the cell proliferation effect of the adipose decellularized matrix freeze-dried composition compared with the cell proliferation effect of the directly freeze-dried composition.

20. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The adipose decellularized matrix freeze-dried composition has one or more characteristics selected from the following group: (1) The reconstitution time of the adipose decellularized matrix freeze-dried composition is 5 to 25 minutes; (2) The stability time of the adipose decellularized matrix freeze-dried composition after reconstitution is greater than 20 minutes; (3) The adipose decellularized matrix freeze-dried composition can be injected through a 30G needle; (4) The cell proliferation effect of the adipose decellularized matrix freeze-dried composition is 10% higher than that of the directly freeze-dried composition.

21. The freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The adipose decellularized matrix freeze-dried composition has one or more characteristics selected from the following group: (1) The reconstitution time of the adipose decellularized matrix freeze-dried composition is 5 to 20 minutes; (2) The stability time of the adipose decellularized matrix freeze-dried composition after reconstitution is greater than 30 minutes; (3) The adipose decellularized matrix freeze-dried composition can be injected through a 32G needle.

22. The method for preparing the freeze-dried adipose decellularized matrix composition according to claim 1, characterized in that: The method comprises the steps of: a. mixing the adipose decellularized matrix with an auxiliary agent to obtain a formulated adipose decellularized matrix emulsion; b. Freeze-drying the prepared adipose decellularized matrix emulsion to obtain the adipose decellularized matrix freeze-dried composition.

23. A use of the freeze-dried adipose decellularized matrix composition according to any one of claims 1 to 21 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 cell proliferation, and the medical cosmetic material is used for plastic surgery filling.

24. A lyophilized powder comprising the lyophilized adipose decellularized matrix composition according to any one of claims 1 to 21.

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