Preparation method of fat acellular extracellular matrix hydrogel

Abstract

The invention belongs to the technical field of biological tissue engineering, and discloses a fat acellular extracellular matrix hydrogel preparation method, which comprises: (1) cutting cleaned fresh fat into pieces, and repeatedly freezing and thawing with a hypertonic salt solution; (2) homogenizing the frozen and thawed fat at low temperature, and collecting and precipitating; (3) re-suspending the precipitate by using PBS, oscillating in a shaking table, centrifuging at room temperature, and collecting the precipitate again; (4) carrying out degreasing and decellularization treatment on the collected precipitate by using a composite eluent; and (5) washing the precipitate with isopropanol for 24 hours, finally repeatedly washing the precipitate with distilled water, and collecting the precipitate, namely the fat acellular matrix. And (6) digesting the fat extracellular matrix with pepsin hydrochloride, and adjusting the pH value to be neutral with sodium hydroxide to obtain the fat extracellular matrix hydrogel. According to the preparation method of the fat extracellular matrix hydrogel, biological enzyme and high-speed centrifugation are not needed in the decellularization stage, the technology is simple, time consumption is short, the yield is high, the fat extracellular matrix prepared through the method is easy to digest, and the prepared hydrogel solution is uniform, good in injectability and capable of being used for production of a filling material for injection.

Description

Technical Field

[0001] This invention belongs to the field of bio-tissue engineering technology, specifically relating to a method for preparing a fat-derived extracellular matrix hydrogel. Background Technology

[0002] Adipose-derived extracellular matrix (DAT) is a bioscaffold obtained by decellularizing and defatting adipose tissue. It retains the components of the natural adipose extracellular matrix, exhibits good biocompatibility and low immunogenicity, and is widely used in tissue engineering. Currently, decellularization methods mainly include: 1. Physical methods such as repeated freeze-thaw cycles, physical compression, and ultrasonic disruption; 2. Chemical methods such as surfactants (sodium dodecyl sulfate, sodium deoxycholate, sodium dodecyl sulfate, sodium dodecyl glutamate, sodium lauryl sulfate, and Triton X-100, Tween-20), acids and alkalis, and hypertonic and hypotonic solutions; 3. Biological methods such as trypsin, nucleases, and ribonucleases. The components retained in scaffolds prepared by different decellularization methods vary, and a single method often yields poor decellularization results. Therefore, the main strategy now is to rationally combine various methods and optimize the preparation process. CN111748526B describes a method where fresh adipose tissue is rapidly minced, treated with 0.25% trypsin-0.1% EDTA and 3% Triton X-100 for 1 hour, followed by treatment with 4% sodium deoxycholate and 100% isopropanol for 1 hour and 6 hours respectively. The trypsin may disrupt the structure of elastin and collagen. CN111603610B describes a method involving three freeze-thaw cycles, washing with hypertonic sodium chloride solution, washing with 1% Triton X-100 solution for 48 hours, isopropanol to precipitate oil, and elution with 75% alcohol three times to obtain the adipose tissue extracellular matrix. CN114286694B uses supercritical fluid solvent extraction to quickly prepare DAT, but requires specialized equipment. CN104877956A describes a more cumbersome process where fresh porcine fat is minced, repeatedly homogenized, shaken, centrifuged, and washed to obtain the adipose tissue extracellular matrix. Animal-derived fats are widely available, but the key to successful preparation lies in removing immunogenic substances while retaining the effective components of the extracellular matrix (ECM) to improve scaffold stability. A common method for preparing decellularized ECM hydrogels is pepsin digestion. This involves adding decellularized ECM powder and pepsin to an acidic solution in a specific ratio, followed by continuous stirring to form a homogeneous ECM digest. The pH and ion concentration of the digest are then adjusted to physiological levels using an alkaline solution and a high-salt buffer to obtain a temperature-sensitive hydrogel. This pre-gel is in a fluid state at 4°C and transforms into a solidified hydrogel at 37°C for 15-30 minutes. The decellularized ECM hydrogel prepared by CN118161667A requires 3 days of digestion at room temperature; the one prepared by CN117258047B requires 72 hours of digestion at room temperature; and the one prepared by CN116850347A requires 48 hours of digestion at 4°C. The digestion time in the ECM hydrogel preparation process is generally long and needs improvement. Therefore, there is an urgent need for a method to prepare fat-derived extracellular matrix hydrogels that can shorten preparation time and improve yield. Summary of the Invention

[0003] To address the problems existing in the prior art, the present invention provides a method for preparing decellularized extracellular matrix from porcine fat, which simplifies the production process, shortens the production time, and improves the yield. The hydrogel prepared by this method is easy to digest and can be used as a filler material for injection.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0005] This invention discloses a method for preparing adipose-derived extracellular matrix, characterized by comprising the following steps: (1) Cut the fresh adipose tissue into small pieces; (2) After shaking the adipose tissue obtained in step (1) in a hypertonic saline solution, replace it with PBS for cyclic freeze-thaw cycles. (3) Add distilled water to the adipose tissue after step (2), homogenize at low temperature, remove the upper layer of coagulated lipids, collect the lower layer of precipitate, and place the precipitate in PBS and shake. (4) Centrifuge the system after shaking in step (3) and collect the lower extracellular matrix precipitate; (5) The precipitate obtained in step (4) is eluted by shaking with a composite eluent containing Tris-HCl, SDS, EDTA, Triton X-100 and Tween 20. (6) Treat the precipitate after elution in step (5) with isopropanol; (7) The precipitate after step (6) is washed, freeze-dried, ground and sterilized to obtain the lipid decellularized extracellular matrix.

[0006] Furthermore, the hypertonic salt solution mentioned in step (2) is a 1M NaCl solution, the shaking treatment temperature is 37℃, and the time is 1h; the cyclic freeze-thaw is performed 3 times.

[0007] Further, in step (3), the amount of distilled water added is 1 / 2 of the volume of the adipose tissue; the homogenate is homogenized at 12000 rpm for 1 min at 4-10℃; and the shaking treatment is carried out at 37℃ for 1 h.

[0008] Furthermore, the centrifugation in step (4) is carried out at 2500-3200 rpm and 25°C for 5 min.

[0009] Further, the composite eluent in step (5) contains 0.1M Tris-HCl, 0.5% SDS, 0.04% EDTA, 1% Triton X-100, 1% Tween 20, and has a pH of 8.8; the elution is carried out with shaking at 37°C for 24 hours.

[0010] This invention also discloses a method for preparing a fat-derived extracellular matrix hydrogel, characterized by comprising the following steps: (a) Take the fat-derived extracellular matrix powder prepared by any of the methods described in claims 1-5; (b) Disperse the powder in a hydrochloric acid solution containing pepsin and digest it by stirring at 4°C; (c) Adjust the pH of the digest solution obtained in step (b) to neutral using sodium hydroxide solution; (d) Add a high-concentration buffer solution to adjust the osmotic pressure of the solution obtained in step (c) to a physiological isotonic state, thereby obtaining the hydrogel.

[0011] Further, in step (b), the concentration of the powder is 10 mg / mL, the concentration of the pepsin is 1 mg / mL, and the concentration of the hydrochloric acid solution is 0.01 M; the digestion time is 24 h.

[0012] The present invention also discloses the application of the hydrogel obtained by the above-described method for preparing fat decellularized extracellular matrix hydrogel in the preparation of injectable soft tissue fillers.

[0013] The present invention also discloses the application of the hydrogel obtained by the above-described method for preparing fat-derived extracellular matrix hydrogel in the preparation of tissue engineering scaffolds.

[0014] The present invention also discloses the application of the hydrogel obtained by the above-described method for preparing fat-derived extracellular matrix hydrogel in the preparation of drug delivery carriers.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] (1) This invention innovatively uses a composite elution solution. Tris buffer provides an alkaline environment, and the anionic surfactant SDS is used in conjunction with the nonionic surfactants Triton X-100 and Tween-20 to improve the efficiency of decellularization and defatting.

[0017] (2) Abundant donor sources.

[0018] (3) No biological enzyme treatment is required in the decellularization stage, which reduces costs and simplifies reagents.

[0019] (4) No need for repeated homogenization and centrifugation, simplifying the process.

[0020] (5) No high-speed centrifuge is required.

[0021] (6) The surfactant concentration is low and relatively mild.

[0022] (7) The substrate is easily digestible and does not require traditional 48-hour digestion.

[0023] (8) The hydrogel has good injectability and can be passed through a 30G needle. Attached Figure Description

[0024] Figure 1 The preparation process of decellularized fat is shown in Figure A. Normal fresh subcutaneous fat; Figure B is an image of cyclic freeze-thaw of adipose tissue; Figure C is a diagram of the low-temperature homogenization process; Figure D is a diagram of the room-temperature shaking process; Figure E is a layered image after centrifugation; Figure F is decellularized fat (DAT) after defatting and decellularization; Figure G is freeze-dried and ground DAT; Figure H is a scanning electron microscope image of DAT.

[0025] Figure 2 The decellularization and defatting effect of fat to remove extracellular matrix.

[0026] Figure 3 These are the results of DNA content detection in normal fat and DAT.

[0027] Figure 4 To prepare and verify the gel properties of fat-derived extracellular matrix hydrogels.

[0028] Figure 5 The images show the injectability of DAT hydrogel, with results obtained using 20G, 22G, 23G, 24G, 27G, and 30G syringe needles. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0030] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0031] Example 1.

[0032] The purpose of this embodiment is to provide a method for preparing fat-derived extracellular matrix hydrogels, the specific steps of which are as follows: 1. Fresh adipose tissue (human, pig, cow, etc.) is rinsed with distilled water and then cut into pieces of 1cm×1cm×1cm.

[0033] 2. Place the adipose tissue in 1M NaCl solution and shake at 37°C for 1 hour, then replace with PBS. Transfer the fat into a container and freeze at -80°C for 24 hours, then thaw in a 37°C water bath. Repeat this freeze-thaw cycle three times.

[0034] 3. Add half the volume of distilled water to the fat mixture, homogenize at 12000 rpm for 1 min, collect the precipitate in PBS and shake at 37°C for 1 h.

[0035] 4. Centrifuge the collected tissue precipitate at 2500-3200 rpm at 25℃ for 5 min. After centrifugation, the material separates into three layers. Collect the white precipitate at the bottom, remove the upper free lipid layer and the middle lipid layer, and collect the lower extracellular matrix precipitate.

[0036] 5. Wash the precipitate with Tris compound eluent at 37℃ with shaking for 24 h, adjust the pH to 8.8 with 0.1M Tris, 0.1M HCl, 0.5% SDS, 0.04% EDTA, 1% Triton X-100, and 1% Tween-20, and wash three times with PBS for 30 min each time.

[0037] 6. Place the precipitate in a 100% isopropanol solution and treat at 37°C for 24 hours to remove residual lipids.

[0038] 7. Wash the above precipitate three times with PBS and three times with distilled water.

[0039] 8. The obtained decellularized fat matrix is ​​freeze-dried, ground into powder at low temperature, and sterilized by cobalt-60 irradiation to obtain sterile decellularized fat extracellular matrix powder, which is stored at -80℃ for long-term use.

[0040] 9. Dissolve the fat-derived extracellular matrix powder prepared by the above method in 10 mg / mL of 0.01 M HCl with 1 mg / mL pepsin and stir at 4°C for 24 h.

[0041] 10. Add 0.1M NaOH to adjust the pH to 7.4.

[0042] 11. Add 10×PBS to adjust to isotonic state.

[0043] Store at 12.4℃ for later use.

[0044] Example 2: Evaluation of decellularization and defatting effects.

[0045] To evaluate the effectiveness of decellularization of fat and removal of extracellular matrix and lipids, this study first prepared frozen sections of fresh adipose tissue and DAT, and then stained them with HE, DAPI and Oil Red O.

[0046] I. Experimental Methods.

[0047] (a) DAT preparation: DAT was prepared according to Example 1.

[0048] (ii) Histological examination.

[0049] 1. OCT embedding and frozen section preparation: Fresh adipose tissue and DAT were embedded in OCT embedding agent, and the section thickness was 8μm. The sections were washed with distilled water for 5 min, fixed with 4% paraformaldehyde for 30 min, then air-dried, labeled and sealed and stored in a -20℃ refrigerator for later use.

[0050] 2. HE Staining Procedure: Hematoxylin-eosin (HE) staining is a commonly used staining method. Hematoxylin staining solution makes the cell nucleus appear blue-purple; eosin stains the cytoplasm and extracellular matrix red. HE staining allows observation of residual cell nuclei after decellularization. The specific staining steps are as follows: (1) Wash the slices with distilled water for 5 minutes; (2) Stain the hematoxylin and chromophores for 5 minutes, then rinse with distilled water; (3) Differentiate with 1% hydrochloric acid-ethanol for 1 second, rinse with tap water and return to blue for 30 minutes; (4) Stain the slide with eosin solution for 1 min, then wash with distilled water for 1 min; (5) Dehydration: 95% ethanol for 5 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min; (6) To achieve transparency: xylene solution I and II, each for 5 minutes; (7) Seal the film with neutral resin, and take pictures for observation after drying.

[0051] 3. Oil Red O Staining Procedure: Oil Red O staining can turn lipid droplets bright red, allowing observation of residual lipid droplets after defatting. First, prepare the working solution: Add saturated Oil Red O stock solution to distilled water at a ratio of 3:2 (Oil Red O: distilled water), mix well, and let stand at room temperature for 10 minutes. Filter using filter paper and a funnel before use. The staining procedure is as follows: (1) Wash the slices with distilled water for 5 minutes; (2) Soak and clean once in 60% isopropanol; (3) Stain with Oil Red O solution for 15 min; (4) 60% isopropanol differentiates to a clear intercellular matrix; (5) Rinse with distilled water; (6) Counterstain cell nuclei with hematoxylin for 3 min; (7) Rinse with distilled water; (8) Glycerin gelatin sealing sheets; (9) Observe under a microscope and take pictures to save the images.

[0052] 4. DAPI staining procedure: DAPI is a common nuclear dye that can turn the cell nucleus blue. The specific steps are as follows: (1) Wash thoroughly with distilled water for 5 minutes to remove the fixative; (2) Place a small amount of DAPI staining solution on the tissue section to cover it, and stain for 5 minutes; (3) After washing with PBS, the slides were mounted. (4) Observe using a fluorescence microscope with a filter wavelength of 360 nm excitation wavelength and 460 nm emission wavelength.

[0053] (iii) Detection of residual DNA.

[0054] Take 25 mg each of lyophilized fresh adipose tissue and DAT (n=3 per group), cut them into small pieces, add 600 μL of Lysis Buffer, and mix thoroughly. Add 20 μL of RNase A, mix well, and let stand at room temperature for 5 min. Add 10 μL of Proteinase K, mix well, and incubate overnight at 56°C. Centrifuge at 12000 rpm / min for 10 min, transfer the supernatant to a new centrifuge tube, add 800 μL of anhydrous ethanol, and mix well. Transfer the liquid to a centrifuge column, centrifuge at 12000 rpm / min for 1 min, and discard the supernatant. Add 700 μL of Washbuffer A (with anhydrous ethanol), centrifuge at 12000 rpm / min for 1 min, and discard the supernatant. Add 700 μL of Wash buffer B (with anhydrous ethanol), centrifuge at 12000 rpm / min for 1 min, and discard the supernatant. Add 500 μL of Wash buffer B, centrifuge at 12000 rpm / min for 1 min, and discard the supernatant. Centrifuge again at 12000 rpm for 2 min. Place the column in a new centrifuge tube, open the column cap, and incubate at 37°C for 10 min, until no obvious ethanol odor remains. Add 50 μL of TE buffer (60°C) to the center of the silica-based membrane, incubate at room temperature for 3 min, centrifuge at 12000 rpm for 30 s, add the supernatant back to a new centrifuge column, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min. The resulting supernatant is the purified genomic DNA. DNA content is determined using an ultra-micro UV spectrophotometer.

[0055] II. Experimental Results.

[0056] like Figure 2 As shown, DAPI staining results revealed scattered blue cell nuclei in normal adipose tissue before decellularization, while no blue fluorescence was visible in the decellularized adipose tissue. HE staining results showed that the DAT group lost the normal structure of adipose tissue, presenting as a disordered fibrous sequence. Microscopically, the filamentous fibers were interwoven, and no blue cell nuclei were visible, confirming the absence of residual cells. Oil Red O staining results showed that no lipid components were visible in the decellularized adipose tissue, proving that defatting was complete.

[0057] The results of DNA quantification showed that ( Figure 3 The DNA content of fresh adipose tissue was 1982.67 ± 17.5 ng / mg, while the DNA content of decellularized adipose tissue was 42.33 ± 18.3 ng / mg. Data are expressed as mean ± standard deviation (n=3), and t-tests were used for comparisons between groups. ***P < 0.001.

[0058] Example 3: Preparation and gel verification of fat-derived extracellular matrix hydrogel.

[0059] I. Experimental Methods.

[0060] (I) Preparation of fat-derived extracellular matrix hydrogel.

[0061] Fat-derived extracellular matrix powder was prepared according to the method described in Example 2 (a), and then hydrogel was prepared according to the following method: (1) Dissolve the fat-derived extracellular matrix powder in 0.01M HCl at a concentration of 10 mg / mL with 1 mg / mL pepsin, and stir at 4°C until the powder is completely dissolved; (2) Add 0.1M NaOH to adjust the pH to 7.4; (3) Add 10×PBS to adjust to isotonic state; (4) Store at 4℃ for later use.

[0062] (II) Verification of the gel properties of decellularized extracellular matrix hydrogels.

[0063] (1) Take 1 mL of the hydrogel prepared in (I) at 4℃ and put it into an EP tube.

[0064] (2) The hydrogel was incubated in a constant temperature incubator at 37°C for 30 minutes.

[0065] (3) Take out the hydrogel and observe the gel condition.

[0066] II. Experimental Results.

[0067] Figure 4 The images show the dissolution state of the decellularized extracellular matrix (ECM) at different digestion times. After 6 hours of digestion, most of the ECM had dissolved, and after 24 hours, almost all of it had dissolved. At 4°C, the ECM hydrogel was a flowing liquid. After incubation at 37°C, the hydrogel changed from a flowing liquid to a non-flowing solid, and its color changed from translucent to milky white, with increased turbidity.

[0068] Example 4: Validation of the injectability of fat-derived extracellular matrix hydrogel.

[0069] I. Experimental Methods.

[0070] (1) Prepare 20G, 22G, 23G, 24G, 27G and 30G syringe needles.

[0071] (2) Remove the original needle from the 1mL syringe, fill it with 1mL of the hydrogel prepared in Example 3 (1), and then add syringe needles of different specifications.

[0072] (3) Gently push the syringe and observe the injectability of the hydrogel.

[0073] II. Experimental Results.

[0074] Figure 5 It can be seen that the decellularized fat extracellular matrix hydrogel can be successfully injected into the needles of 20G, 22G, 23G, 24G, 27G and 30G syringes, indicating that the decellularized fat extracellular matrix hydrogel prepared by this method has good injectability.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing fat-derived extracellular matrix, characterized in that, Includes the following steps: (1) Cut the fresh adipose tissue into small pieces; (2) After shaking the adipose tissue obtained in step (1) in a hypertonic saline solution, replace it with PBS for cyclic freeze-thaw cycles. (3) Add distilled water to the adipose tissue after step (2), homogenize at low temperature, remove the upper layer of coagulated lipids, collect the lower layer of precipitate, and place the precipitate in PBS and shake. (4) Centrifuge the system after shaking in step (3) and collect the lower extracellular matrix precipitate; (5) The precipitate obtained in step (4) is eluted by shaking with a composite eluent containing Tris-HCl, SDS, EDTA, Triton X-100 and Tween 20. (6) Treat the precipitate after elution in step (5) with isopropanol; (7) The precipitate after step (6) is washed, freeze-dried, ground and sterilized to obtain the lipid decellularized extracellular matrix.

2. The method according to claim 1, characterized in that, The hypertonic salt solution mentioned in step (2) is a 1M NaCl solution, the shaking treatment temperature is 37℃, and the time is 1 hour; the cyclic freeze-thaw is performed 3 times.

3. The method according to claim 1, characterized in that, In step (3), the amount of distilled water added is 1 / 2 of the volume of the adipose tissue; the homogenate is homogenized at 12000 rpm for 1 minute at 4-10℃; and the shaking treatment is carried out at 37℃ for 1 hour.

4. The method according to claim 1, characterized in that, The centrifugation in step (4) is carried out at 2500-3200 rpm and 25°C for 5 minutes.

5. The method according to claim 1, characterized in that, The composite eluent in step (5) contains 0.1 M Tris-HCl, 0.5% SDS, 0.04% EDTA, 1% Triton X-100, 1% Tween 20, and has a pH of 8.8; the elution is carried out with shaking at 37°C for 24 hours.

6. A method for preparing a fat-derived extracellular matrix hydrogel, characterized in that, Includes the following steps: (a) Take the fat-derived extracellular matrix powder prepared by any of the methods described in claims 1-5; (b) Disperse the powder in a hydrochloric acid solution containing pepsin and digest it by stirring at 4°C; (c) Adjust the pH of the digest solution obtained in step (b) to neutral using sodium hydroxide solution; (d) Add a high-concentration buffer solution to adjust the osmotic pressure of the solution obtained in step (c) to a physiological isotonic state, thereby obtaining the hydrogel.

7. The method according to claim 6, characterized in that, In step (b), the concentration of the powder is 10 mg / mL, the concentration of the pepsin is 1 mg / mL, and the concentration of the hydrochloric acid solution is 0.01 M; the digestion time is 24 hours.

8. The application of the hydrogel obtained by the method for preparing fat-derived extracellular matrix hydrogel according to claim 6 in the preparation of injectable soft tissue fillers.

9. The application of the hydrogel obtained by the method for preparing fat-derived extracellular matrix hydrogel according to claim 6 in the preparation of tissue engineering scaffolds.

10. The application of the hydrogel obtained by the method for preparing fat-derived extracellular matrix hydrogel according to claim 6 in the preparation of drug delivery carriers.

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