Novel 34G fat product, preparation method and application

Abstract

The invention provides a preparation method of a novel 34G fat product, which comprises the following steps: S1, cleaning lidocaine, epinephrine medicine, red blood cells, grease and cell debris in a fat suction substance, and removing impurities; s2, performing chylification operation on the washed fat, performing chylification for 12-24 times back and forth, and controlling the time to be within 20-30 minutes; s3, enabling the chylified fat to pass through a sieve of 60 to 100 meshes; s4, putting the fat screened by the screen into an injector, and pushing the fat to pass through a 26-29G needle; and S5, sucking fat into an injector connected with a 26-29G needle, replacing the injector with a 34G needle, and pushing out the fat of the 34G needle to obtain the novel 34G fat product. The fat product provided by the invention contains fat cells, fat stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, progenitor cells and growth factors and proteins secreted by various fat, and can be used for beautifying and treating various skin diseases with non-treatment purposes.

Description

Technical Field

[0001] This patent relates to the application of a new type of 34G fat product in technical fields such as regenerative medicine and cosmetic surgery, and specifically to a new type of 34G fat product, preparation method and application. Background Art

[0002] Fat transplantation technology has a history of more than 100 years. Ordinary fat transplantation uses granular fat, which can be used to fill soft tissue depressions and have a certain regenerative effect on the skin of the treatment area, such as reducing wrinkles, promoting blood circulation, reducing fibrosis, immune regulation, and reducing pigmentation. However, traditional fat is in a granular semi-solid and semi-liquid form and is usually used for injection into the subcutaneous fat layer or the periosteum surface. Injection into the shallow layer will result in uneven granularity.

[0003] Nano fat transplantation is the same as ordinary fat transplantation. It first extracts fat tissue from the abdomen or thighs and other parts by negative pressure suction, and then centrifuges the fat to obtain adipose tissue with high purity. After emulsifying and filtering these pure fat tissues, nano fat is obtained. Conventional fat transplantation contains a large number of fat cells, while nano fat does not have fat cells, but it has abundant fat precursor stem cells. It is these fat precursor stem cells that play the role of filling depressions and promoting tissue regeneration and rejuvenation. Conventional fat transplantation contains large pieces of fat tissue, so it needs to be performed with a thicker fat injection needle. It can only fill larger depressions or tissue capacity loss caused by fat or muscle atrophy, such as filling the temples, forehead, apple muscles, nose dorsum, eye bags, etc., and cannot be injected into the dermis. Nano fat particles are extremely small and can be directly injected with a very fine needle, such as a needle for hyaluronic acid, which can directly fill the atrophy of the dermis.

[0004] However, even the nano fat in the existing technology can only pass through 25 or 27G needles, and the fat cells are removed from the nano fat, which will cause nodules and vascular embolism after injection. Summary of the invention

[0005] The present invention aims to solve the problems of insufficient injection accuracy, easy generation of nodules and vascular embolism in the existing fat transplantation technology, and provides a new 34G fat product and its preparation method. This product not only contains cell groups including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, and progenitor cells, but is also rich in a variety of growth factors and proteins secreted by fat, and can achieve precise injection through a 34G needle. It is particularly suitable for applications in soft tissue repair and remodeling, skin texture improvement, and facial rejuvenation. At the same time, it can also be used for filling treatment of local defects, improvement of scars and wrinkles, and promotion of chronic wound healing and repair of refractory wounds. Through these applications, the new 34G fat product has demonstrated broad potential and practical value in multiple medical and cosmetic fields.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing a novel 34G fat product, comprising the following steps:

[0008] S1. Clean lidocaine, adrenaline, red blood cells, oil and cell debris in the liposuctioned material and remove impurities therein;

[0009] S2, emulsifying the washed fat for 12-24 times, each emulsification time being 20-30 minutes;

[0010] S3, filtering the emulsified fat through a 60-100 mesh sieve;

[0011] S4, put the sieved fat into the syringe and push the fat through the 26-29G needle;

[0012] S5. Use a syringe connected to a 26-29G needle to absorb fat, then replace it with a 34G needle. The fat pushed out of the 34G needle is the prepared new 34G fat product.

[0013] Preferably, in step S1, the lidocaine, adrenaline, red blood cells, lipids and cell debris in the lipoaspirate are washed multiple times with a single or a combination of isotonic solutions such as physiological saline, PBS buffer, Ringer's solution or glucose solution, and impurities are removed by low-speed centrifugation.

[0014] Preferably, in step S3, the mesh size of the sieve used is specifically 60-100 meshes.

[0015] Preferably, in step S1, the volume ratio of the lipoaspirate to the isotonic solution during washing is 1:1.

[0016] Preferably, a method for preparing a novel 34G fat product comprises the following steps:

[0017] S1. Washing lidocaine, adrenaline drugs, red blood cells, fat and cell fragments in the lipoaspirate: Use a single or combination of isotonic solutions such as physiological saline, PBS buffer, Ringer's solution or glucose solution to wash lidocaine, adrenaline drugs, red blood cells, fat and cell fragments in the lipoaspirate multiple times to remove impurities such as drugs, blood, fat and cell fragments attached to the fat surface. During the washing process, the volume ratio of the lipoaspirate to the isotonic solution is preferably 1:1, and the adipose tissue is further purified by low-speed centrifugation (such as 64g, 1 minute).

[0018] S2. Emulsification: The cleaned fat is placed in an emulsifier (including electric or manual types) for emulsification. The emulsification times are 12-24 times, and each emulsification time is 20-30 minutes to ensure that the fat tissue is evenly and appropriately refined.

[0019] S3, filtration treatment: the emulsified fat is filtered through a 60-100 mesh sieve to remove larger particles and inadequately emulsified fat tissue to ensure the fineness of the final product;

[0020] S4, refinement: put the sieved fat into a syringe and push the fat through a 26-29G needle to further reduce the size of fat particles;

[0021] S5. Preparation of 34G fat product: Use a syringe connected to a 26-29G needle to absorb the refined fat, then replace it with a 34G needle and slowly push out the fat to obtain the novel 34G fat product of the present invention.

[0022] The second aspect of the present invention provides a novel 34G fat product, which includes a cell population including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, progenitor cells, and growth factors and proteins secreted by fat, and the product is prepared by the method described in the present invention.

[0023] Preferably, the growth factor is one or a combination of VEGF, HGF, IL-6, EGF, FGF, NGF, IGF, PDGF, EPO, and TGF.

[0024] In the present invention, the growth factors and proteins secreted by fat give the product unique biological activity and significant therapeutic effect. The product is obtained by the above-mentioned carefully designed preparation method, which ensures the integrity of the ingredients and the uniformity of the product.

[0025] The third aspect of the present invention provides a cell population including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells and progenitor cells separated from a novel 34G fat product. The cell population separated from the novel 34G fat product is obtained by collagenase treatment, hypoxic environment culture and laser irradiation technology.

[0026] Preferably, the growth factor is one or a combination of VEGF, HGF, IL-6, EGF, FGF, NGF, IGF, PDGF, EPO, and TGF.

[0027] In the present invention, the collagen network in the novel 34G fat product is destroyed by the enzymatic hydrolysis of collagenase, so that cell groups including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, and progenitor cells are more easily released while maintaining the integrity and activity of the cells. It is particularly suitable for precise filling of small areas such as crow's feet.

[0028] In the present invention, through the low oxygen environment culture technology, cell groups including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, and progenitor cells can better adapt to the new growth environment, promote cell survival and proliferation, while maintaining their differentiation potential, and provide a high-quality cell source for subsequent filling or therapeutic applications.

[0029] In the present invention, laser irradiation can promote the metabolic activities of cell groups including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, and progenitor cells, enhance their biological activities, and may promote the secretion of cytokines, providing additional biological effects for filling or treatment.

[0030] The fourth aspect of the present invention provides an application of a novel 34G fat product in medical treatment and cosmetic surgery for non-therapeutic purposes, wherein soft tissue filling, shaping or regenerative medicine is performed using the novel 34G fat product of the present invention or cell groups including fat cells, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells and progenitor cells separated from the novel 34G fat product of the present invention.

[0031] The fifth aspect of the present invention provides an application of a novel 34G fat product in wound healing or skin regeneration treatment for non-therapeutic purposes, wherein the novel 34G fat product of the present invention or a cell population including fat cells, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, and progenitor cells separated from the novel 34G fat product of the present invention are injected into a wound site or an area requiring skin regeneration to promote tissue repair and regeneration.

[0032] The sixth aspect of the present invention provides an application of a novel 34G fat product in cell therapy or tissue engineering for non-therapeutic purposes, using the novel 34G fat product of the present invention or a cell population including fat cells, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, and progenitor cells separated from the novel 34G fat product of the present invention as a cell source or tissue scaffold to construct or repair human tissues.

[0033] Compared with the prior art, the beneficial effects and significant improvements of the technical solution of the present invention are:

[0034] 1. Rich ingredients: The new 34G fat product provided by the present invention contains cell groups including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, progenitor cells, and various factors and proteins secreted by fat (HGF protein, IL-6 protein, etc.), providing multiple mechanism support for treatment;

[0035] 2. High injection accuracy: 34G needle achieves precise injection and is suitable for small areas.

[0036] 3. High safety: avoid the use of chemical substances such as emulsifiers to reduce the risk of fat damage.

[0037] 4. Significant effect: It shows excellent therapeutic effect in facial filling and skin repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention are briefly introduced below.

[0039] Figure 1 This is an electron microscope scanning image of a new type of 34G fat product in Example 3;

[0040] Figure 2 The results of osteogenic differentiation experiment in Example 3, wherein 2A is the blank control group, 2B is the staining picture of the sample after 14 days of induction, and 2C is the staining picture of the sample after 19 days of induction;

[0041] Figure 3 The adipogenic differentiation experiment results of Example 3, wherein 3A is the blank control group, and 3B is the staining image of the sample after 14 days of induction;

[0042] Figure 4 The results of the chondrogenic differentiation experiment in Example 3, wherein 4A is the spherical cartilage in a centrifuge tube, 4B is the cartilage after induction observed under a microscope, and 4C is the cartilage after being made into a pressed sheet;

[0043] Figure 5 This is a picture showing the results of changing the medium on the third day of culture in Example 3;

[0044] Figure 6 This is a picture showing the results of changing the medium on the 6th day of culture in Example 3;

[0045] Figure 7 This is a picture showing the results of changing the medium on the 9th day of culture in Example 3;

[0046] Figure 8 This is a picture showing the results of taking a photo of the medium change on the 13th day of culture in Example 3. DETAILED DESCRIPTION

[0047] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0048] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the application.

[0049] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0050] Emulsification refers to the mechanical or chemical treatment of adipose tissue to make it more delicate and homogeneous and possibly change its physical or chemical properties. Emulsification usually involves the use of specific equipment or chemical reagents to break up fat cells, resulting in a more delicate, injectable fat emulsion. This process helps to improve the usability of adipose tissue, especially in medical and cosmetic applications such as soft tissue augmentation and body shaping.

[0051] The 34g needle has a willow-leaf-shaped blade design at the tip, which can further reduce penetration resistance and provide a guarantee for painless injection. The 34g needle is applicable to type A pen syringes and is used for subcutaneous injections of drugs including insulin and growth hormone. It can effectively help diabetic patients relieve pain and discomfort during self-injection.

[0052] According to international standards, the sizes and specifications of injection needles in China are: the inner diameter of a 23g needle is 0.317mm, the inner diameter of a 25g needle is 0.232mm, the inner diameter of a 26g needle is 0.232mm, the inner diameter of a 27g needle is 0.184mm, the inner diameter of a 28g needle is 0.133mm, the inner diameter of a 29g needle is 0.133mm, and the inner diameter of a 34g needle is 0.080mm.

[0053] The experimental materials and basic experimental methods used in the following examples are briefly introduced as follows:

[0054] 1. Experimental Materials

[0055] ●Lipoaspirate: obtained from healthy volunteers, approved by the ethics committee.

[0056] ●Cleaning solution: physiological saline, PBS buffer, Ringer's solution, glucose solution (all are isotonic solutions).

[0057] ●Emulsifier: electric emulsifier and manual emulsifier.

[0058] ●Screen: 60 mesh, 80 mesh, 100 mesh, 110 mesh stainless steel screen.

[0059] ●Syringe and needle: 1mL syringe, 26-29G and 34G needle.

[0060] ●Other reagents: Trypan blue, red blood cell lysis buffer, flow cytometry antibodies (CD31, CD34, CD45, CD44, CD73, CD90), Alizarin red, Oil red O, Alcian blue, etc.

[0061] 2. Experimental Methods

[0062] ● Fat pretreatment: Use isotonic solution (normal saline, PBS buffer, Ringer's solution or glucose solution) in a 1:1 volume ratio to wash lidocaine, adrenaline drugs, red blood cells, lipids and cell debris in the lipoaspirate multiple times, and remove impurities by low-speed centrifugation (64g, 1 minute).

[0063] ●Emulsion treatment: Place the cleaned fat in an emulsifier and perform emulsion treatment for different times and durations according to the experimental design.

[0064] ●Filtration: Use sieves of different mesh sizes to filter the emulsified fat.

[0065] ●Thinning process: The filtered fat is thinned through a 26-29G needle.

[0066] ●Preparation of 34G fat products: Use a syringe connected to a 26-29G needle to absorb the refined fat, and then replace it with a 34G needle to push out the fat product.

[0067] ●Product testing: including electron microscope scanning, fat yield calculation, trypan blue counting, flow cytometric analysis, three-lineage differentiation detection, protein factor detection and culture status observation, etc.

[0068] Example 1 Emulsification screening experiment

[0069] Experimental groups with different creaming times and times were designed to screen out the optimal creaming conditions.

[0070] Group 1

[0071] 1.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat, cell debris and other substances;

[0072] 1.2. Take 40 mL of fat and use the emulsifier to emulsify it 16 times continuously for 20 minutes;

[0073] 1.3. Pass the emulsified fat through an 80-mesh stainless steel sieve;

[0074] 1.4. Take the fat after the sieve and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0075] 1.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle. The fat product can be slowly pushed out of the needle.

[0076] Group 2

[0077] 2.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris;

[0078] 2.2. Take 40 mL of fat and use an electric emulsifier to emulsify it 20 times continuously for 25 minutes;

[0079] 2.3. Pass the emulsified fat through an 80-mesh stainless steel sieve;

[0080] 2.4. Take the fat after the mesh and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0081] 2.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle. The fat product can be slowly pushed out of the needle.

[0082] Group 3

[0083] 3.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris;

[0084] 3.2. Take 40 mL of fat and use an electric emulsifier to emulsify it 24 times continuously for 30 minutes;

[0085] 3.3. Pass the emulsified fat through an 80-mesh stainless steel sieve;

[0086] 3.4. Take the fat after the sieve and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0087] 3.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle. The fat product can be slowly pushed out of the needle.

[0088] Group 4 (lack of chylolysis)

[0089] 4.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris;

[0090] 4.2. Take 40 mL of fat and use an electric emulsifier to emulsify it 12 times continuously for 15 minutes;

[0091] 4.3. Pass the emulsified fat through an 80-mesh stainless steel sieve. Most of the fat cannot pass through the 80-mesh stainless steel sieve.

[0092] 4.4. Take a small amount of fat after passing through the mesh and put it into a 1mL syringe. Push the fat through the original needle (0.45mm*16mm) connected to the 1mL syringe and push it into another centrifuge tube.

[0093] 4.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle. The fat product cannot be pushed out of the needle.

[0094] Group 5 (over-chylolysis)

[0095] 5.1. Fat pretreatment: Use normal temperature saline in a 1:1 ratio to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris.

[0096] 5.2. Take 40 mL of fat and use an electric emulsifier to emulsify it 28 times continuously for 35 minutes;

[0097] 5.3. Pass the emulsified fat through an 80-mesh stainless steel sieve;

[0098] 5.4. Take the fat after the mesh and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0099] 5.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, change to a 34G needle, and the fat product can be slowly pushed out of the needle. However, under the microscope, the fat cells of the fat product in group 5 were severely damaged, the number of cells was small, and the cell activity was reduced.

[0100] Conclusion: The results show that 16-24 times of creaming, 20-30 minutes each time, combined with 80-mesh sieve filtration, can produce a fat product suitable for passing through a 34G needle. Insufficient or excessive creaming will affect product quality.

[0101] Example 2 Screening Experiment

[0102] Group 6

[0103] 6.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris;

[0104] 6.2. Take 40 mL of fat and use an electric emulsifier to emulsify it 16 times continuously for 20 minutes;

[0105] 6.3. Pass the emulsified fat through a 60-mesh stainless steel sieve;

[0106] 6.4. Take the fat after the mesh and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0107] 6.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle.

[0108] Fat products can be slowly pushed out of the needle, but there is greater resistance to pushing.

[0109] Group 7

[0110] 7.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris;

[0111] 7.2. Take 40 mL of fat and use an electric emulsifier to emulsify it 16 times continuously for 20 minutes;

[0112] 7.3. Pass the emulsified fat through a 100-mesh stainless steel sieve;

[0113] 7.4. Take the fat after the mesh and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0114] 7.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle.

[0115] The fat product can be slowly pushed out of the needle.

[0116] Group 8 (large aperture mesh screen)

[0117] 8.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris;

[0118] 8.2. Take 40 mL of fat and emulsify it 16 times using an electric emulsifier for 20 minutes;

[0119] 8.3. Pass the emulsified fat through a 50-mesh stainless steel sieve;

[0120] 8.4. Take the fat after the sieve and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0121] 8.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle. The fat product cannot be pushed out of the needle.

[0122] Group 9 (Small aperture mesh screen)

[0123] 9.1. Fat pretreatment: Use normal temperature saline at a ratio of 1:1 to wash lidocaine, adrenaline, red blood cells, fat and cell debris in the liposuctioned material for multiple times, and centrifuge at low speed (64g for 1 minute) to remove blood, fat and cell debris;

[0124] 9.2. Take 40 mL of fat and use an electric emulsifier to emulsify it 16 times continuously for 20 minutes;

[0125] 9.3. Pass the emulsified fat through a 110-mesh stainless steel sieve. Only a small amount of fat can pass through the 110-mesh stainless steel sieve, and most of the fat is intercepted on the stainless steel sieve;

[0126] 9.4. Take the fat after the sieve and put it into a 1mL syringe. Push the fat through a 26-29G needle and push it into another centrifuge tube.

[0127] 9.5. Use a 1mL syringe connected to a 26-29G needle to draw fat into the syringe, then change to a 34G needle.

[0128] The fat product can be slowly pushed out of the needle.

[0129] Based on the above experimental results, it can be seen that in the method of the present invention, a 60-100 mesh screen can produce qualified 34G fat products. If the mesh screen aperture is too large, the fat will not be able to be pushed out from the 34G needle; if the mesh screen aperture is too small, most of the fat will be intercepted by the screen, resulting in excessive loss.

[0130] Conclusion: By comparing the filtration effects of sieves with different mesh sizes, it is determined that the 60-100 mesh sieve is the best choice. Too large or too small an aperture is not conducive to the preparation of qualified 34G fat products.

[0131] Example 3 Basic Tests of New 34G Fat Products

[0132] 3.1 Scanning electron microscope

[0133] The fat product of group 1 was placed under an electron microscope. The results were as follows Figure 1 As shown, it can be seen that the new 34G fat product contains a complete cell structure.

[0134] 3.2 Fat Yield

[0135] In group 1, 40 ml of washed fat can yield 30 ml of 34G fat product, with a fat yield of 75%.

[0136] 3.3. Trypan blue counting

[0137] In trypan blue counting group 1, the number of cells contained in 34G fat was 2.2×105 / ml, and the cell viability was 80.5%; while the number of cells contained in normal fat was 1×105~1*106 / ml, and the cell viability was 79.1%, which was close to the cell number and cell viability after conventional fat treatment.

[0138] 3.4 Streaming Analysis

[0139] The cells obtained by digesting 34 g of fat in Group 1 and normal fat (complete cells but with more impurities) were all centrifuged and resuspended in 1 mL of PBS. The flow cytometry results showed (the results are shown in Table 1) that the 34 g fat product of the present invention was similar to normal fat in cell type and content, but had fewer impurities.

[0140] Table 1

[0141]

[0142]

[0143] 3.5 Three-lineage differentiation

[0144] The cells treated with 34g fat product from group 1 were cultured adherently, and the obtained cells were subjected to a three-lineage differentiation test. The results are shown in Table 1. Figure 2 , Figure 3 and Figure 4 The results show that the 34G fat prepared by the present invention has the ability of three-lineage differentiation of mesenchymal stem cells after culture.

[0145] 3.6 Protein factor detection

[0146] The fat products in Group 1 were tested for HGF and IL-6, respectively. The results showed that the expression levels of HGF and IL-6 protein factors in the 34G fat of the present invention and the liposuction material were similar, and the activity was well retained. The HGF test results are shown in Tables 2 and 3, and the IL-6 test results are shown in Tables 4 and 5.

[0147] Table 2

[0148]

[0149] Table 3

[0150]

[0151] Table 4

[0152]

[0153]

[0154] Table 5

[0155]

[0156] 3.7. Training

[0157] The fat products of group 1 were cultured and observed under a microscope on the 3rd, 6th, 9th and 13th days of culture. Figure 5-8 As shown, 5 is the photographic result of changing the medium on the 3rd day of culture; Figure 6 This is the picture showing the results of changing the medium on the 6th day of culture; Figure 7 This is a picture of the results of changing the medium on the 9th day of culture; Figure 8 The following is a picture of the results of changing the medium on the 13th day of culture. Figure 5-8 As shown, with the extension of culture time, the cell morphology gradually changed into spindle-shaped fibroblast-like cells, indicating that the cell activity was good.

[0158] Example 4 Facial nasolabial groove filling experiment

[0159] 1. Conventional method for facial nasolabial fold filling

[0160] Liposuction: Use tumescent anesthesia technology in obese areas such as the waist, abdomen, thighs, buttocks, etc., and use liposuction equipment to suck out the subcutaneous fat needed for transplantation.

[0161] Fat particle purification: The aspirated fat particles are processed through fat purification technology to extract the fat particles needed for injection.

[0162] Fat injection: Inject purified fat particles into the skin at the outer edge of the orbit, and perform massage, shaping and appropriate pressure bandage after the operation.

[0163] In conventional methods, fat particles prepared from autologous fat are used to fill the nasolabial folds on the face. The fat particles do not contain fat cells and adipose stem cells, but are mainly composed of components such as extracellular matrix. The volume retention rate after surgery is high, but because the internal fibrous tissue is still not removed, it can only pass through a 27G needle, which is prone to form cord-like nodules.

[0164] 2. The method of the present invention is used to fill the facial nasolabial groove

[0165] When the fat product of Example 1 is used to fill the nasolabial folds on the face, the surgical operator can use a 34G needle for filling. The amount of fat filled is controllable and cord-like nodules are less likely to form.

[0166] Comparison of the effects of conventional fat particle filling and the 34G fat product of the present invention. The results show that the skin surface of the facial nasolabial fold area filled with the method of the present invention is more full, and the long-term effect is better (no nasolabial fold depression reappeared after 18 months). The reason may be that the fat cells retained in the product maintain the vitality of the implanted site.

[0167] The applicant declares that, in the description of the above specification:

[0168] The descriptions of the terms "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0169] Finally, it should be noted that:

[0170] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0171] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims

1. A method for preparing a novel 34G fat product, characterized in that: The following steps are involved: S1. Clean lidocaine, adrenaline, red blood cells, oil and cell debris in the liposuctioned material and remove impurities therein; S2, emulsifying the washed fat for 12-24 times, each emulsification time being 20-30 minutes; S3, filtering the emulsified fat through a 60-100 mesh sieve; S4. Place the sieved fat into a syringe and push the fat through a 26-29G needle. S5. Use a syringe connected to a 26-29G needle to absorb the emulsified fat, then replace it with a 34G needle. The fat pushed out of the 34G needle is the prepared new 34G fat product.

2. The method for preparing the novel 34G fat product according to claim 1, characterized in that: In step S1, the lidocaine, adrenaline, red blood cells, lipids and cell debris in the lipoaspirate are washed multiple times with a single or a combination of isotonic solutions such as physiological saline, PBS buffer, Ringer's solution or glucose solution, and impurities are removed by low-speed centrifugation.

3. The method for preparing the novel 34G fat product according to claim 1, characterized in that: In step S3, the mesh size of the sieve used is specifically 60-100 meshes.

4. The method for preparing the novel 34G fat product according to claim 2, characterized in that: In step S1, the volume ratio of the lipoaspirate to the isotonic solution during washing is 1:

1.

5. A new type of 34G fat product, characterized in that: The product comprises cell groups including adipocytes, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, progenitor cells, and growth factors and proteins secreted by fat, and the product is prepared by the method described in any one of claims 1 to 4.

6. A cell population isolated from the novel 34G fat product according to claim 5, characterized in that: The cell population separated from the novel 34G fat product is obtained by collagenase treatment, low oxygen environment culture and laser irradiation technology.

7. The growth factor secreted by the novel 34G fat product according to claim 5 or the novel 34G fat product according to claim 6, characterized in that: The growth factor is one or a combination of VEGF, HGF, IL-6, EGF, FGF, NGF, IGF, PDGF, EPO and TGF.

8. Application of a novel 34G fat product in non-therapeutic medical and cosmetic surgery, characterized in that: Soft tissue filling, shaping or regenerative medicine is performed according to the new 34G fat product according to claim 5 or the cell population including fat cells, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, progenitor cells separated from the new 34G fat product according to claim 6, and the growth factors and proteins secreted by fat.

9. A novel 34G fat product for use in wound healing or skin regeneration treatment for non-therapeutic purposes, characterized in that: The cell populations including fat cells, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, progenitor cells separated from the new 34G fat product according to claim 5 or the new 34G fat product according to claim 6, as well as growth factors and proteins secreted by fat, are injected into the wound site or the area requiring skin regeneration to promote tissue repair and regeneration.

10. Application of a novel 34G fat product in cell therapy or tissue engineering for non-therapeutic purposes, characterized in that: The cell populations including fat cells, adipose stem cells, endothelial cells, smooth muscle cells, fibroblasts, immune cells, and progenitor cells separated from the novel 34G fat product described in claim 5 or the novel 34G fat product described in claim 6, as well as growth factors and proteins secreted by fat are used as cell sources or tissue scaffolds to construct or repair human tissues.

Images