Method for preparing acellular fat matrix
The method of two trypsin treatments and neutral cleaning solutions effectively prepares an acellular fat matrix with improved biocompatibility and mechanical properties, addressing the limitations of existing DAT preparation methods.
Patent Information
- Application Number
- CN202411425506.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-12
- Publication Date
- 2024-12-27
AI Technical Summary
Existing methods for preparing decellularized adipose tissue (DAT) are inadequate in completely removing cell components, leading to immunogenicity, cytotoxicity, and reduced biocompatibility, which affects the efficacy of soft tissue repair and wound healing.
A method involving two trypsin treatments and extraction processes, combined with the use of neutral cleaning solutions like Na2HPO4 and KH2PO4, and the addition of phenylmethylsulfonyl fluoride to preserve the extracellular matrix structure, is employed to prepare acellular fat matrix.
The method results in a decellularized fat matrix that is clean, non-immunogenic, and retains bioactive components, enhancing its biocompatibility and mechanical properties, making it effective for soft tissue repair and wound healing.
Smart Images
Figure CN119185660A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medical biomaterials, and in particular to a method for preparing acellular fat matrix. Background Art
[0002] Soft tissue defects include defects of skin, fat, muscle and other connective tissues, which are mainly caused by trauma, tumor resection, aging and other surgeries. These injuries are often too large to heal normally on their own, and the patient's quality of life declines due to loss of function, and it also imposes a huge economic burden on the healthcare system. Skin flap or myocutaneous flap transplantation and fat transplantation are usually used clinically to solve contour abnormalities and soft tissue defects. However, the transplantation of skin flaps and myocutaneous flaps has the disadvantages of donor site abnormalities, loss of function and transplantation failure. Although adipose tissue has the advantages of easy access and rich in adipose-derived stem cells (ASCs), fat transplantation has certain limitations. For example, the survival rate and volume filling effect of autologous adipose tissue transplantation are unpredictable. Autologous adipose tissue needs to be extracted from the body, fat preservation is difficult, and the source of some individuals is limited. Allogeneic fat transplantation has problems such as immunogenicity.
[0003] Decellularized adipose tissue (DAT) is an extracellular matrix without cell components obtained by effectively removing immunogenic cell components from adipose tissue through a series of physical / chemical / biological treatments. DAT has good biocompatibility and almost no tissue rejection and allergic reactions even when transplanted allogeneically. DAT is rich in collagen and glycosaminoglycan (GAG), which can simulate the natural cell microenvironment, support the directional induction and regeneration of tissues such as fat, blood vessels and nerves, and play an important regulatory role in cell adhesion, migration, proliferation, differentiation and gene expression, especially stem cell proliferation and lineage-specific differentiation. Decellularized matrix can retain its inherent shape and conformation, and can also be processed into new structures. Extracellular matrix materials that retain their original structure provide a complete vascular system, an accurate model of its derived tissues, and good mechanical strength.
[0004] Traditional methods for preparing DAT include physical method, chemical method, bioenzyme method and combined method. Physical methods mainly include freeze-thaw method, high water pressure method and supercritical carbon dioxide method. Although physical methods are relatively mild, can retain the effective components of decellularized matrix and cause less damage to physical structure, they cannot completely remove cell components and genetic material when used alone and need to be used in combination with other methods. Chemical methods achieve decellularization by dissolving the phospholipid components of cell membranes. Commonly used chemical reagents include sodium dodecyl sulfate (SDS), sodium deoxycholate (SD) and Triton-100. Chemical reagents can achieve strong decellularization, but they are easy to remain or agglomerate on decellularized materials. In order to reduce the impact of residues, additional washing time is required, which not only reduces the decellularization efficiency, but also reduces the content of GAG and growth factors during repeated washing, affecting the adhesion and growth ability of cells. Bioenzyme method uses the hydrolysis of enzymes to remove cell components or cut the peptide bonds connecting cells and matrix. Commonly used biological enzymes include trypsin, ribonucleases, deoxyribonucleases, and lipases. Trypsin can be used directly to remove cellular components from tissues, and the effect will increase over time, but long-term exposure to trypsin will reduce the content of elastin and GAG in the decellularized matrix. RNases and deoxyribonucleases are often used in combination with chemical reagents SDS or SD, which helps to further hydrolyze nuclear materials after the chemical reagents destroy the cell membrane and release genetic material, but RNases and deoxyribonucleases will damage the surface structure of DAT and affect cell adhesion and proliferation.
[0005] Therefore, optimizing the DAT preparation process to obtain a DAT that is more completely decellularized, non-immunogenic, non-cytotoxic, and has good biocompatibility is an urgent problem to be solved by those skilled in the art. Summary of the invention
[0006] In view of the above-mentioned defects of the prior art, the present invention provides a method for preparing acellular fat matrix, and the technical problem to be solved is to more completely retain the components of the extracellular matrix.
[0007] In order to solve the above problems, the technical solution adopted by the present invention is: a method for preparing acellular fat matrix, the method comprising the following steps:
[0008] 1) Providing adipose tissue raw materials;
[0009] 2) subjecting the adipose tissue raw material to freeze-thaw treatment to obtain adipose tissue after freeze-thaw treatment;
[0010] 3) performing a first trypsin treatment on the adipose tissue after the freeze-thaw treatment to remove cells, thereby obtaining adipose tissue after the first trypsin treatment;
[0011] 4) subjecting the adipose tissue after the first trypsin treatment to a first extraction treatment, washing, and draining to obtain the adipose tissue after the first extraction treatment;
[0012] 5) subjecting the adipose tissue after the first extraction treatment to a second trypsin treatment to further remove cells, washing, and draining to obtain the adipose tissue after the second trypsin treatment;
[0013] 6) hydrolyzing the adipose tissue after the second trypsin treatment, washing, and draining to obtain adipose tissue after hydrolysis treatment;
[0014] 7) The hydrolyzed fat tissue is subjected to a second extraction treatment, washed, and drained to obtain the decellularized fat matrix.
[0015] Preferably, in steps 4), 5), 6) and 7), the cleaning solution used for the cleaning treatment contains Na2HPO4, KH2PO4, phenylmethylsulfonyl fluoride, penicillin and streptomycin.
[0016] Preferably, in step 1), the step of providing adipose tissue raw material comprises: placing the obtained adipose tissue in sterile PBS supplemented with 1%-2% bovine serum albumin, and transporting it in a refrigerated manner.
[0017] Preferably, in step 2), the freeze-thaw solution used in the freeze-thaw treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or
[0018] In steps 3) and 5), the trypsin treatment solution used in the first trypsin treatment and the second trypsin treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or
[0019] In steps 4) and 7), the extracts used in the first extraction treatment and the second extraction treatment further contain phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or
[0020] In step 6), the hydrolysis solution used for the hydrolysis treatment also contains phenylmethylsulfonyl fluoride, penicillin and streptomycin.
[0021] Preferably, in step 2), the freeze-thaw treatment comprises: freezing below -80°C for 1 to 2 hours, then heating to 25 to 37°C, and repeating 3 to 5 times.
[0022] Preferably, the first trypsin treatment and the second trypsin treatment are performed at a temperature of 25 to 37° C. for 8 to 16 hours.
[0023] Preferably, in steps 4) and 7), the extracting liquid used in the first extraction process and the second extraction process contains isopropanol.
[0024] Preferably, in steps 4) and 7), the first extraction treatment and the second extraction treatment are carried out at 25-37° C. for 8-16 hours.
[0025] Preferably, in step 6), the hydrolysis solution used in the hydrolysis treatment contains deoxyribonuclease, ribonuclease and lipase.
[0026] Preferably, in step 6), the hydrolysis treatment is carried out at 25-37° C. for 16-24 h.
[0027] Preferably, in step 7), the acellular fat matrix is obtained and subjected to freeze-drying treatment.
[0028] The beneficial effects of the present invention are as follows: compared with the traditional method, the method for preparing the decellularized fat matrix provided by the present invention is clean and free of impurities, has excellent physical properties, good mechanical properties, can better retain the components of the extracellular matrix intact, has significant biocompatibility, low immunogenicity, and can retain more bioactive components (mainly cytokines) to be more conducive to exerting biological functions, can be better applied to repair soft tissue defects and promote wound healing, and is expected to provide a new strategy for the treatment of soft tissue defects and chronic wound healing on the basis of establishing a new DAT process.
[0029] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 This is a photograph of the appearance of the decellularized fat matrix prepared in Example 1.
[0031] Figure 2 These are stained micrographs of sections of adipose tissue (AT) and decellularized adipose matrix (DAT) prepared in Example 1.
[0032] Figure 3 This is a scanning electron microscope photograph of the decellularized fat matrix prepared in Example 1. DETAILED DESCRIPTION
[0033] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0034] The present invention has two main innovations compared to the prior art. The first innovation is that the method for preparing acellular fat matrix provided by the present invention includes two trypsin treatments and extraction treatments. When the inventors of the present application were exploring the preparation method, they initially used one trypsin treatment and extraction treatment. After many experiments, adjusting various parameters, and using various reagents, they found that the effect of removing cells and fat was not good, especially the effect of removing fat was not ideal. In one experiment, due to an erroneous operation, two trypsin treatments and extraction treatments were performed, and it was unexpectedly found that the effect of removing cells and fat was particularly good. The second innovation is the cleaning solution. The present invention adopts neutral and milder cleaning solutions such as Na2HPO4 and KH2PO4 to wash away the precipitated fat and cells in the tissue, thereby better preserving the structure of the extracellular matrix. In addition, the present invention creatively adds phenylmethylsulfonyl fluoride to the cleaning solution. Phenylmethylsulfonyl fluoride is a good protease inhibitor, which can ensure that the protein in the extracellular matrix remains active from the time it is retrieved from the tissue to the end of the preparation, and does not deteriorate or denature, thereby better preserving the structure of the extracellular matrix. Furthermore, the inventors of the present application also found that adding an antibacterial agent to the cleaning solution can prevent bacteria from multiplying in the tissue and promoting protein denaturation, thereby better preserving the structure of the extracellular matrix.
[0035] Example 1
[0036] Prepare the solution:
[0037] Prepare phenylmethylsulfonyl fluoride (PMSF) solution: add 25 mL of isopropanol to 1 g of PMSF and stir until transparent.
[0038] Prepare freeze-thaw solution: weigh 1.21 g Tris and 1.46 g EDTA, add deionized water to a final volume of 1 L and stir until dissolved, adjust the pH to 7.0, add 5 μL PMSF solution and 1 mL penicillin-streptomycin solution (100× double antibody).
[0039] Prepare trypsin treatment solution: first prepare 0.25% trypsin-EDTA solution, then add 5 μL PMSF solution and 1 mL 100× double antibody solution to every 100 ml trypsin-EDTA solution.
[0040] Prepare the extraction solution: add 5 μL PMSF solution and 1 mL 100× double antibody solution per 100 ml isopropanol.
[0041] Prepare cleaning solution: weigh 19.6392 g Na2HPO4·12H2O and 2.3 g KH2PO4, add deionized water to a final volume of 1 L and stir until dissolved, adjust the pH to 7.0, add 5 μL PMSF solution and 1 mL 100× double antibody solution.
[0042] Prepare NaCl-glycerol mixture: weigh 0.175 g NaCl and dissolve it in 10 mL deionized water, adjust the pH to 7.3, add 10 mL glycerol and mix thoroughly.
[0043] Prepare DNase stock solution: Add 15 mL of NaCl-glycerol mixture to 200,000 units of DNase and mix.
[0044] Prepare Tris-NaCl solution: weigh 0.06 g Tris and 0.44 g NaCl and dissolve in 50 mL deionized water, and adjust the pH to 7.5.
[0045] Prepare RNase stock solution: add 40 mL Tris-NaCl solution to 500 mg RNase.
[0046] Prepare frozen CaCl2 solution: weigh 0.02775 g of CaCl2 powder, add deionized water to a final volume of 50 mL, dissolve thoroughly, and cool to 4°C.
[0047] Prepare lipase stock solution: add 50 mL of chilled CaCl2 solution to 100,000 units of lipase.
[0048] Prepare hydrolyzate: weigh 19.6392 g Na2HPO4·12H2O, 0.58536 g MgSO4 and 2.3 g KH2PO4, add deionized water to a final volume of 1 L and stir until dissolved, adjust the pH to 7.0, and add 1 mL of deoxyribonuclease stock solution, 1 mL of ribonuclease stock solution, 2 mL of lipase stock solution, 5 μL of PMSF solution and 1 mL of 100× double antibody solution for every 200 ml of liquid.
[0049] (1) Acquisition and preservation of adipose tissue raw materials
[0050] Adipose tissue should be obtained from people aged 18-50 years old. Before obtaining adipose tissue, the donor should be screened for a history of tuberculosis, hepatitis, syphilis, AIDS, tumors, malaria, and leprosy. At the same time, it should be ensured that the donor has no large-area burn lesions, no autoimmune diseases, no use of steroid drugs for no more than three months, no history of drug or other substance poisoning, and no radiation therapy in the donor area. HIV-1 / 2-Ab, HBs-Ag, HCV-Ab, RPR, and HBc-Ab in the donor serum test report must be negative. After the fat resection surgery, adipose tissue is obtained in a clean environment and placed in sterile PBS supplemented with 1% bovine serum albumin (BSA) to minimize tissue loss and obtain a higher yield of decellularized matrix. The tissue is packaged with disinfected or sterilized tools or containers, and the acquisition process is registered. During transportation, the tissue is stored in a refrigerated transport box at 4°C. When the tissue is received, the donor's medical history and test results must be verified, and the time, quantity, type, and transportation of the tissue acquisition must be registered. The tissue should be frozen in a 4℃ refrigerator for no more than 72 hours; the tissue can be stored in a -80℃ refrigerator for long-term storage. This is the fat tissue raw material.
[0051] As an alternative to this specific implementation, the above adipose tissue may also be lipoaspirate.
[0052] (2) Freeze-thaw treatment
[0053] Use forceps and surgical scissors to remove the sterile fat tissue from the fat tissue raw material obtained in step (1), transfer it to a new sterile specimen jar, add freeze-thaw solution at a volume ratio of 1:1, freeze at -80°C for 1 hour, then heat to 25°C, replace the freeze-thaw solution after it is fully thawed, repeat 3 times, and finally pour out the freeze-thaw solution to obtain the fat tissue after freeze-thaw treatment. The fat tissue after freeze-thaw treatment melts and softens.
[0054] This freeze-thaw solution makes it easier to remove oils and fats, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0055] (3) First treatment with trypsin
[0056] The freeze-thawed adipose tissue obtained in step (2) was added with trypsin treatment solution at a volume ratio of 1:1, and stirred for digestion: temperature 25°C, frequency 50 rpm, time 8 hours, and the trypsin treatment solution was removed to obtain the adipose tissue after the first trypsin treatment.
[0057] This trypsin treatment can reduce the activity of proteases released during cell lysis while removing cells, thereby retaining cytokines.
[0058] (4) Extraction first treatment
[0059] The trypsin-treated adipose tissue obtained in step (3) was added to the extract at a volume ratio of 1:1, and stirred continuously at 25° C. with a frequency of 50 rpm. The extract was replaced twice during the stirring period for 8 hours.
[0060] After the extraction is completed, the extract is removed, and then the fat tissue is washed and drained to obtain the fat tissue after the first extraction treatment.
[0061] The specific steps of washing and draining are: adding washing liquid at a volume ratio of 1:1, stirring continuously at 25°C, a frequency of 50 rpm, a time of 20 minutes, repeating 3 times, and draining the water;
[0062] With this extraction process, isopropanol is miscible in water and oil and is used to extract lipids from tissues, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0063] (5) Second trypsin treatment
[0064] The adipose tissue after the first extraction treatment obtained in step (4) is subjected to a second trypsin treatment by the same method as step (3), and then washed and drained by the same method as step (4) to obtain the adipose tissue after the second trypsin treatment.
[0065] (6) Hydrolysis treatment
[0066] The trypsin-treated adipose tissue obtained in step (5) was added to the hydrolyzate at a volume ratio of 1:1, and the mixture was stirred continuously at 25° C., with a frequency of 50 rpm, for 16 h. The hydrolyzate was removed, and then the adipose tissue was washed and drained in the same manner as in step (4) to obtain the hydrolyzed adipose tissue.
[0067] This hydrolysis treatment can further remove fat and residual nucleic acids, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0068] (7) Extraction Second Treatment
[0069] The hydrolyzed adipose tissue obtained in step (6) was extracted by the same method as step (4), washed and drained, and then rinsed in 60% ethanol for 20 minutes, repeated 3 times, and then freeze-dried to obtain acellular adipose matrix. The freeze-drying process is as follows: the sample is placed in a centrifuge tube and frozen at -20°C overnight. The sample is freeze-dried for 48 hours.
[0070] (8) Properties of the prepared decellularized fat matrix
[0071] (a) Appearance observation of the prepared decellularized fat matrix
[0072] The appearance of the prepared decellularized fat matrix is shown in the attached figure. Figure 1As shown, it can be seen that the DAT tissue has a light yellow-white appearance (darker yellow or brown appearance indicates incomplete extraction of lipids and / or cells), is clean and free of impurities. After removal, it was found that the physical properties and mechanical properties were excellent.
[0073] (b) Histological characteristics of the prepared decellularized adipose tissue matrix
[0074] Adipose tissue (AT) and prepared decellularized adipose matrix (DAT) were cryosectioned and stained with HE, Masson, Oil Red O and DAPI. The micrographs are shown in Figure 2 As shown in the figure. As can be seen from the figure, HE staining results show that DAT has no cell structure, only red-stained extracellular matrix components can be seen, and a grid-like scaffold microstructure can be seen inside. Masson staining results show that there is no cell structure in DAT, only blue-stained collagen fiber structure can be seen. Oil Red O staining results only show the scaffold structure of DAT, without red-stained lipid droplets and blue-stained cell nuclei. DAPI staining results show that there is no blue fluorescent cell nuclear component in DAT.
[0075] The scanning electron microscopy images of the prepared decellularized adipose tissue matrix (DAT) are shown in Figure 2. Figure 3 As shown in the figure, it can be seen that there is no cell structure in DAT, which presents a porous structure with different pore sizes and obvious collagen fiber entanglement.
[0076] (c) DNA content of the prepared decellularized fat matrix
[0077] In order to evaluate the effect of decellularization during preparation, the DNA content in the sample was measured using the magnetic bead capture method. Three batches of adipose tissue were prepared for DAT using the above protocol, and the residual DNA was detected. The results are shown in Table 1 below.
[0078] Table 1. Results of residual DNA content determination of DAT
[0079]
[0080] As can be seen from the table, the residual DNA levels of the three batches were 18.9 ng / mg, 13.37 ng / mg and 8.32 ng / mg, respectively, which met Gilbert's evaluation criteria for DNA content in decellularized tissues of <50 ng / mg dry weight, indicating that there was almost no residual DNA in DAT and that it was non-immunogenic.
[0081] Example 2
[0082] Prepare the solution:
[0083] Prepare phenylmethylsulfonyl fluoride (PMSF) solution: add 60 mL of isopropanol to 2 g of PMSF and stir until transparent.
[0084] Prepare freeze-thaw solution: weigh 1.21 g Tris and 1.46 g EDTA, add deionized water to a final volume of 2 L and stir until dissolved, adjust the pH to 8.0, add 15 μL PMSF solution and 2 mL penicillin-streptomycin solution (100× double antibody).
[0085] Prepare trypsin treatment solution: first prepare 0.05% trypsin-EDTA solution, then add 15 μL PMSF solution and 2 mL 100× double antibody solution to every 100 ml trypsin-EDTA solution.
[0086] Prepare the extraction solution: add 15 μL PMSF solution and 2 mL 100× double antibody solution to every 100 ml isopropanol.
[0087] Prepare cleaning solution: weigh 19.6392 g Na2HPO4·12H2O and 2.3 g KH2PO4, add deionized water to a final volume of 2 L and stir until dissolved, adjust the pH to 8.0, add 15 μL PMSF solution and 2 mL 100× double antibody solution.
[0088] Prepare NaCl-glycerol mixture: weigh 0.175 g NaCl and dissolve it in 10 mL deionized water, adjust the pH to 7.3, add 10 mL glycerol and mix thoroughly.
[0089] Prepare DNase stock solution: Add 15 mL of NaCl-glycerol mixture to 200,000 units of DNase and mix.
[0090] Prepare Tris-NaCl solution: weigh 0.06 g Tris and 0.44 g NaCl and dissolve in 50 mL deionized water, and adjust the pH to 7.5.
[0091] Prepare RNase stock solution: add 40 mL Tris-NaCl solution to 500 mg RNase.
[0092] Prepare frozen CaCl2 solution: weigh 0.02775 g of CaCl2 powder, add deionized water to a final volume of 50 mL, dissolve thoroughly, and cool to 4°C.
[0093] Prepare lipase stock solution: add 50 mL of chilled CaCl2 solution to 100,000 units of lipase.
[0094] Prepare hydrolyzate: weigh 19.6392 g Na2HPO4·12H2O, 0.58536 g MgSO4 and 2.3 g KH2PO4, add deionized water to a final volume of 2 L and stir until dissolved, adjust the pH to 8.0, and add 2 mL of deoxyribonuclease stock solution, 2 mL of ribonuclease stock solution, 4 mL of lipase stock solution, 15 μL of PMSF solution and 2 mL of 100× double antibody solution for every 200 ml of liquid.
[0095] (1) Acquisition and preservation of adipose tissue raw materials
[0096] Adipose tissue should be obtained from people aged 18-50 years old. Before obtaining adipose tissue, the donor should be screened for a history of tuberculosis, hepatitis, syphilis, AIDS, tumors, malaria, and leprosy. At the same time, it should be ensured that the donor has no large-area burn lesions, no autoimmune diseases, no use of steroid drugs for no more than three months, no history of drug or other substance poisoning, and no radiation therapy in the donor area. HIV-1 / 2-Ab, HBs-Ag, HCV-Ab, RPR, and HBc-Ab in the donor serum test report must be negative. After the fat resection surgery, adipose tissue is obtained in a clean environment and placed in sterile PBS supplemented with 2% bovine serum albumin (BSA) to minimize tissue loss and obtain a higher yield of decellularized matrix. The tissue is packaged with disinfected or sterilized tools or containers, and the acquisition process is registered. During transportation, the tissue is stored in a refrigerated transport box at 4°C. When the tissue is received, the donor's medical history and test results must be verified, and the time, quantity, type, and transportation of the tissue acquisition must be registered. The tissue should be frozen in a 4℃ refrigerator for no more than 72 hours; the tissue can be stored in a -80℃ refrigerator for long-term storage. This is the fat tissue raw material.
[0097] As an alternative to this specific implementation, the above adipose tissue may also be lipoaspirate.
[0098] (2) Freeze-thaw treatment
[0099] Use forceps and surgical scissors to remove sterile fat tissue from the fat tissue raw material obtained in step (1), transfer it to a new sterile specimen jar, add freeze-thaw solution at a volume ratio of 1:2, freeze at -80°C for 2 hours, then heat to 37°C, replace the freeze-thaw solution after it is fully thawed, repeat 5 times, and finally pour out the freeze-thaw solution to obtain the fat tissue after freeze-thaw treatment. The fat tissue after freeze-thaw treatment melts and softens.
[0100] This freeze-thaw solution makes it easier to remove oils and fats, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0101] (3) First treatment with trypsin
[0102] The freeze-thawed adipose tissue obtained in step (2) was added with trypsin treatment solution at a volume ratio of 1:2, and stirred for digestion at a temperature of 37° C., a frequency of 150 rpm, and a time of 16 h. The trypsin treatment solution was removed to obtain adipose tissue after the first trypsin treatment.
[0103] This trypsin treatment can reduce the activity of proteases released during cell lysis while removing cells, thereby retaining cytokines.
[0104] (4) Extraction first treatment
[0105] The trypsin-treated adipose tissue obtained in step (3) was added to the extract at a volume ratio of 1:2, and stirred continuously at 37°C at a frequency of 150 rpm. The extract was replaced 3 times during the period of 16 hours.
[0106] After the extraction is completed, the extract is removed, and then the fat tissue is washed and drained to obtain the fat tissue after the first extraction treatment.
[0107] The specific steps of washing and draining are as follows: adding washing solution at a volume ratio of 1:2, stirring continuously at 37°C, a frequency of 150 rpm, a time of 30 min, repeating 5 times, and draining the water;
[0108] With this extraction process, isopropanol is miscible in water and oil and is used to extract lipids from tissues, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0109] (5) Second trypsin treatment
[0110] The adipose tissue after the first extraction treatment obtained in step (4) is subjected to a second trypsin treatment by the same method as step (3), and then washed and drained by the same method as step (4) to obtain the adipose tissue after the second trypsin treatment.
[0111] (6) Hydrolysis treatment
[0112] The trypsin-treated adipose tissue obtained in step (5) was added with hydrolysis solution at a volume ratio of 1:2, and stirred continuously at 37° C., with a frequency of 150 rpm, for 24 h, and the hydrolysis solution was removed. The adipose tissue was then washed and drained in the same manner as in step (4) to obtain hydrolyzed adipose tissue.
[0113] This hydrolysis treatment can further remove fat and residual nucleic acids, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0114] (7) Extraction Second Treatment
[0115] The hydrolyzed adipose tissue obtained in step (6) was extracted by the same method as step (4), washed and drained, and then rinsed in 80% ethanol for 30 minutes, repeated 5 times, and then freeze-dried to obtain acellular adipose matrix. The freeze-drying process is as follows: the sample is placed in a centrifuge tube and frozen at -80°C overnight. The sample is freeze-dried for 72 hours.
[0116] (8) Properties of the prepared decellularized fat matrix
[0117] Through appearance observation, the acellular fat matrix prepared in this example has the same light yellow-white appearance as that in Example 1, is clean and free of impurities, has excellent physical properties, and has good mechanical properties.
[0118] The decellularized fat matrix prepared in this example was sectioned and stained by the same method as in Example 1, and the same histological characteristics as those prepared in Example 1 were found. The HE staining results showed that DAT had no cell structure, and only red-stained extracellular matrix components were visible, and a grid-like scaffold microstructure was visible therein. The Masson staining results showed that there was no cell structure in DAT, and only blue-stained collagen fiber structure was seen. The Oil Red O staining results showed that only the scaffold structure of DAT was visible, and no red-stained lipid droplets and blue-stained cell nuclei were seen. The DAPI staining results showed that there were no blue fluorescent cell nuclear components in DAT.
[0119] The decellularized fat matrix prepared in this example was observed by scanning electron microscopy using the same method as in Example 1, and the same histological characteristics as those of the decellularized fat matrix prepared in Example 1 were found. The DAT had no cell structure, and presented a porous structure with different pore sizes, with obvious collagen fiber entanglements.
[0120] The same method as in Example 1 was used to measure the DNA content of the decellularized fat matrix prepared in this example. The result was similar to that in Example 1, which met the evaluation criteria proposed by Gilbert that the DNA content in decellularized tissue was less than 50 ng / mg dry weight, indicating that there was almost no residual DNA in the DAT and it was non-immunogenic.
[0121] Example 3
[0122] Prepare the solution:
[0123] Prepare phenylmethylsulfonyl fluoride (PMSF) solution: add 40 mL of isopropanol to 2 g of PMSF and stir until transparent.
[0124] Prepare freeze-thaw solution: weigh 1.21 g Tris and 1.46 g EDTA, add deionized water to a final volume of 1.5 L and stir until dissolved, adjust the pH to 7.5, add 10 μL PMSF solution and 1.5 mL penicillin-streptomycin solution (100× double antibody).
[0125] Prepare trypsin treatment solution: first prepare 0.125% trypsin-EDTA solution, and then add 10 μL PMSF solution and 1.5 mL 100× double antibody solution to every 100 ml trypsin-EDTA solution.
[0126] Prepare the extraction solution: add 10 μL PMSF solution and 1.5 mL 100× double antibody solution per 100 mL isopropanol.
[0127] Prepare cleaning solution: weigh 19.6392 g Na2HPO4·12H2O and 2.3 g KH2PO4, add deionized water to a final volume of 1.5 L and stir until dissolved, adjust the pH to 7.5, add 10 μL PMSF solution and 1.5 mL 100× double antibody solution.
[0128] Prepare NaCl-glycerol mixture: weigh 0.175 g NaCl and dissolve it in 10 mL deionized water, adjust the pH to 7.3, add 10 mL glycerol and mix thoroughly.
[0129] Prepare DNase stock solution: Add 15 mL of NaCl-glycerol mixture to 200,000 units of DNase and mix.
[0130] Prepare Tris-NaCl solution: weigh 0.06 g Tris and 0.44 g NaCl and dissolve in 50 mL deionized water, and adjust the pH to 7.5.
[0131] Prepare RNase stock solution: add 40 mL Tris-NaCl solution to 500 mg RNase.
[0132] Prepare frozen CaCl2 solution: weigh 0.02775 g of CaCl2 powder, add deionized water to a final volume of 50 mL, dissolve thoroughly, and cool to 4°C.
[0133] Prepare lipase stock solution: add 50 mL of chilled CaCl2 solution to 100,000 units of lipase.
[0134] Prepare hydrolyzate: weigh 19.6392 g Na2HPO4·12H2O, 0.58536 g MgSO4 and 2.3 g KH2PO4, add deionized water to a final volume of 1.5 L and stir until dissolved, adjust the pH to 8.5, and add 1.5 mL of deoxyribonuclease stock solution, 1.5 mL of ribonuclease stock solution, 3 mL of lipase stock solution, 10 μL of PMSF solution and 1.5 mL of 100× double antibody solution for every 200 ml of liquid.
[0135] (1) Acquisition and preservation of adipose tissue raw materials
[0136] Adipose tissue should be obtained from people aged 18-50 years old. Before obtaining adipose tissue, the donor should be screened for a history of tuberculosis, hepatitis, syphilis, AIDS, tumors, malaria, and leprosy. At the same time, it should be ensured that the donor has no large-area burn lesions, no autoimmune diseases, no use of steroid drugs for no more than three months, no history of drug or other substance poisoning, and no radiation therapy in the donor area. HIV-1 / 2-Ab, HBs-Ag, HCV-Ab, RPR, and HBc-Ab in the donor serum test report must be negative. After the fat resection surgery, adipose tissue is obtained in a clean environment and placed in sterile PBS supplemented with 1.5% bovine serum albumin (BSA) to minimize tissue loss and obtain a higher yield of decellularized matrix. The tissue is packaged with disinfected or sterilized tools or containers, and the acquisition process is registered. During transportation, the tissue is stored in a refrigerated transport box at 4°C. When the tissue is received, the donor's medical history and test results must be verified, and the time, quantity, type, and transportation of the tissue acquisition must be registered. The tissue should be frozen in a 4℃ refrigerator for no more than 72 hours; the tissue can be stored in a -80℃ refrigerator for long-term storage. This is the fat tissue raw material.
[0137] As an alternative to this specific implementation, the above adipose tissue may also be lipoaspirate.
[0138] (2) Freeze-thaw treatment
[0139] Use forceps and surgical scissors to take out the sterile fat tissue from the fat tissue raw material obtained in step (1), transfer it to a new sterile specimen jar, add freeze-thaw solution at a volume ratio of 1:1.5, freeze at -80°C for 1.5 hours, then heat to 37°C, replace the freeze-thaw solution after it is fully thawed, repeat 4 times, and finally pour out the freeze-thaw solution to obtain the fat tissue after freeze-thaw treatment. The fat tissue after freeze-thaw treatment melts and softens.
[0140] This freeze-thaw solution makes it easier to remove oils and fats, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0141] (3) First treatment with trypsin
[0142] The freeze-thawed adipose tissue obtained in step (2) was added with trypsin treatment solution at a volume ratio of 1:1.5, and stirred for digestion: temperature 37°C, frequency 100 rpm, time 10 h, and the trypsin treatment solution was removed to obtain the adipose tissue after the first trypsin treatment.
[0143] This trypsin treatment can reduce the activity of proteases released during cell lysis while removing cells, thereby retaining cytokines.
[0144] (4) Extraction first treatment
[0145] The trypsin-treated adipose tissue obtained in step (3) was added to the extract at a volume ratio of 1:1.5, and stirred continuously at 37° C. with a frequency of 100 rpm. The extract was replaced 3 times during the period, and the time was 10 hours.
[0146] After the extraction is completed, the extract is removed, and then the fat tissue is washed and drained to obtain the fat tissue after the first extraction treatment.
[0147] The specific steps of washing and draining are: adding washing solution at a volume ratio of 1:1.5, stirring continuously at 37°C, frequency of 100 rpm, time of 25 minutes, repeating 4 times, and draining the water;
[0148] With this extraction process, isopropanol is miscible in water and oil and is used to extract lipids from tissues, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0149] (5) Second trypsin treatment
[0150] The adipose tissue after the first extraction treatment obtained in step (4) is subjected to a second trypsin treatment by the same method as step (3), and then washed and drained by the same method as step (4) to obtain the adipose tissue after the second trypsin treatment.
[0151] (6) Hydrolysis treatment
[0152] The trypsin-treated adipose tissue obtained in step (5) was added with hydrolyzate at a volume ratio of 1:1.5, and stirred continuously at 37° C., with a frequency of 100 rpm, for 20 h, and the hydrolyzate was removed. The adipose tissue was then washed and drained in the same manner as in step (4) to obtain hydrolyzed adipose tissue.
[0153] This hydrolysis treatment can further remove fat and residual nucleic acids, reduce the activity of proteases released during cell lysis, and retain cytokines.
[0154] (7) Extraction Second Treatment
[0155] The hydrolyzed adipose tissue obtained in step (6) was extracted by the same method as step (4), washed and drained, and then rinsed in 70% ethanol for 25 minutes, repeated 4 times, and then freeze-dried to obtain acellular adipose matrix. The freeze-drying process is as follows: the sample is placed in a centrifuge tube and frozen at -80°C overnight. The sample is freeze-dried for 60 hours.
[0156] (8) Properties of the prepared decellularized fat matrix
[0157] Through appearance observation, the acellular fat matrix prepared in this example has the same light yellow-white appearance as that in Example 1, is clean and free of impurities, has excellent physical properties, and has good mechanical properties.
[0158] The decellularized fat matrix prepared in this example was sectioned and stained by the same method as in Example 1, and the same histological characteristics as those prepared in Example 1 were found. The HE staining results showed that DAT had no cell structure, and only red-stained extracellular matrix components were visible, and a grid-like scaffold microstructure was visible therein. The Masson staining results showed that there was no cell structure in DAT, and only blue-stained collagen fiber structure was seen. The Oil Red O staining results showed that only the scaffold structure of DAT was visible, and no red-stained lipid droplets and blue-stained cell nuclei were seen. The DAPI staining results showed that there were no blue fluorescent cell nuclear components in DAT.
[0159] The decellularized fat matrix prepared in this example was observed by scanning electron microscopy using the same method as in Example 1, and the same histological characteristics as those of the decellularized fat matrix prepared in Example 1 were found. The DAT had no cell structure, and presented a porous structure with different pore sizes, with obvious collagen fiber entanglements.
[0160] The same method as in Example 1 was used to measure the DNA content of the decellularized fat matrix prepared in this example. The result was similar to that in Example 1, which met the evaluation criteria proposed by Gilbert that the DNA content in decellularized tissue was less than 50 ng / mg dry weight, indicating that there was almost no residual DNA in the DAT and it was non-immunogenic.
[0161] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A method for preparing acellular fat matrix, characterized in that: The method comprises the following steps: 1) Providing adipose tissue raw materials; 2) subjecting the adipose tissue raw material to freeze-thaw treatment to obtain adipose tissue after freeze-thaw treatment; 3) performing a first trypsin treatment on the adipose tissue after the freeze-thaw treatment to remove cells, thereby obtaining adipose tissue after the first trypsin treatment; 4) subjecting the adipose tissue after the first trypsin treatment to a first extraction treatment, washing, and draining to obtain the adipose tissue after the first extraction treatment; 5) subjecting the adipose tissue after the first extraction treatment to a second trypsin treatment to further remove cells, washing, and draining to obtain the adipose tissue after the second trypsin treatment; 6) hydrolyzing the adipose tissue after the second trypsin treatment, washing, and draining to obtain adipose tissue after hydrolysis treatment; 7) The hydrolyzed fat tissue is subjected to a second extraction treatment, washed, and drained to obtain the decellularized fat matrix.
2. The method according to claim 1, characterized in that In steps 4), 5), 6) and 7), the cleaning solution used for the cleaning treatment contains Na2HPO4, KH2PO4, phenylmethylsulfonyl fluoride, penicillin and streptomycin.
3. The method according to claim 1, characterized in that In step 2), the freeze-thaw solution used in the freeze-thaw treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or In steps 3) and 5), the trypsin treatment solution used in the first trypsin treatment and the second trypsin treatment contains phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or In steps 4) and 7), the extracts used in the first extraction treatment and the second extraction treatment further contain phenylmethylsulfonyl fluoride, penicillin and streptomycin; and / or In step 6), the hydrolysis solution used for the hydrolysis treatment also contains phenylmethylsulfonyl fluoride, penicillin and streptomycin.
4. The method according to claim 1, characterized in that In step 2), the freeze-thaw treatment includes: freezing below -80°C for 1 to 2 hours, then heating to 25 to 37°C, and repeating 3 to 5 times.
5. The method according to claim 1, characterized in that The first trypsin treatment and the second trypsin treatment are carried out at a temperature of 25 to 37° C. for 8 to 16 hours.
6. The method according to claim 1, characterized in that In steps 4) and 7), the extracting solution used in the first extraction process and the second extraction process contains isopropyl alcohol.
7. The method according to claim 1, characterized in that In steps 4) and 7), the first extraction treatment and the second extraction treatment are carried out at 25-37° C. for 8-16 hours.
8. The method according to claim 1, characterized in that In step 6), the hydrolysis solution used for the hydrolysis treatment contains deoxyribonuclease, ribonuclease and lipase.
9. The method according to claim 1, characterized in that In step 6), the hydrolysis treatment is carried out at 25-37° C. for 16-24 h.
10. The method according to claim 1, characterized in that In step 7), the acellular fat matrix is obtained and subjected to freeze-drying treatment.