Method for preparing umbilical cord acellular matrix, umbilical cord acellular matrix and use thereof

Abstract

The application discloses a preparation method of umbilical cord decellularized matrix based on perfusion decellularization technology, which comprises decellularization treatment of a complete umbilical cord by using a programmed perfusion method, and cleaning, enzymatic hydrolysis, freeze-drying and other processes. The application further discloses the umbilical cord decellularized matrix prepared by the method and application thereof. The obtained umbilical cord decellularized matrix has complete structural integrity, retains not only umbilical cord Wharton's jelly but also rich low-immunogenic extracellular matrix components in umbilical cord tissues, and has wide application prospects in the development of soft tissue injury repair, medical beauty and life beauty related products.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials technology and medical aesthetics, specifically to a method for preparing decellularized umbilical cord matrix, the decellularized matrix, and its uses. Background Technology

[0002] Given the crucial role of stem cells and their bioactive substances in skin repair, the demand for stem cell-based beauty products is increasing daily. The beauty industry is exploring the use of mesenchymal stem cells isolated from newborn umbilical cords and adult adipose tissue; however, stem cell technology faces several potential challenges regarding stability. Currently, adding one or a few active substances to beauty products is insufficient to achieve skin repair and anti-aging effects. Therefore, simulating the complex microenvironment necessary for stem cell growth may become a new trend in beauty products.

[0003] Animal-derived biological raw materials are being widely used in the development of bioproducts. Among them, it is well known that the umbilical cord contains abundant mesenchymal stem cells, suggesting its potential as a human stem cell replacement product. In addition, umbilical cord walnut gel is rich in natural components such as collagen, elastin, hyaluronic acid, and growth factors, which can also help mimic the complex microenvironment necessary for stem cell growth, making it suitable for skin care and repair.

[0004] However, the umbilical cord contains a large amount of heterologous antigens, which can easily trigger an immune response. Due to the evolutionary conservation of extracellular matrix components among different mammals, it inherently possesses low immunogenicity. Therefore, decellularization techniques can be used to prepare low-immunogenic umbilical cord matrix extracts, but conventional elution methods easily cause damage to the umbilical cord structure and loss of Wharton's jelly.

[0005] Preparing decellularized matrix from whole organs via vascular perfusion is another major decellularization method besides tissue block elution. It utilizes only a single artery / venous vessel entering / exiting the organ as the perfusion pathway. Through the capillary network within the solid organ and the closed loop formed between the arteries and veins, it allows for deeper and more uniform penetration into the tissue. Decellularization conditions are gentler, with less matrix loss, reduced decellularization time, and improved decellularization efficiency. However, the umbilical cord contains one umbilical vein and two umbilical arteries. Due to the vascular distribution characteristics of the umbilical cord, an internal circulation cannot be formed between the umbilical vein and arteries, which hinders the preparation of umbilical cord matrix extracts using the aforementioned traditional vascular perfusion methods. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing decellularized umbilical cord matrix that can effectively preserve the extracellular matrix components of umbilical cord cells.

[0007] According to one aspect of the present invention, a method for preparing decellularized umbilical cord matrix is ​​provided, comprising the following steps: cleaning the umbilical cord to remove blood stains from the surface of the umbilical cord; perfusing the umbilical cord vessels with physiological saline and / or phosphate buffer to remove blood from the umbilical cord; perfusing decellularized fluid through the umbilical cord vessels to perform programmed elution of cells in the umbilical cord; and perfusing phosphate buffer through the umbilical cord vessels to remove residual decellularized fluid from the umbilical cord, thereby obtaining decellularized umbilical cord matrix.

[0008] In some implementations, optionally, the obtained decellularized umbilical cord matrix is ​​cut, freeze-dried, and ground to obtain a freeze-dried powder of decellularized umbilical cord matrix.

[0009] In some embodiments, optionally, the decellularized umbilical cord matrix lyophilized powder is weighed, enzymatically hydrolyzed in a protease solution, and the pH value is adjusted to obtain a decellularized umbilical cord matrix solution. In some embodiments, optionally, the obtained decellularized umbilical cord matrix solution is lyophilized again to obtain a secondary lyophilized umbilical cord matrix powder.

[0010] In some implementations, the umbilical cord is derived from a human or a non-human mammal.

[0011] In some embodiments, the umbilical cord vessels are one or more of two umbilical arteries and one umbilical vein; preferably, they are perfused via one umbilical vein and at least one umbilical artery; more preferably, they are perfused via one umbilical vein and two umbilical arteries.

[0012] In some embodiments, the decellularized fluid perfused via the umbilical cord blood vessels preferably contains one or more of the following: 0.1% to 5.0% (w / v) Triton X-100, 0.1% to 5.0% (w / v) sodium dodecyl sulfate, and 100 to 5000 U / L of DNase; more preferably, a gradient of decellularized fluids is used for sequential perfusion.

[0013] The gradient can be a gradient consisting of decellularization solution I and decellularization solution II. The components of decellularization solution I can be: sodium dodecyl sulfate at a concentration of 1.5–3.5% (w / v), DNase at a concentration of 3500–4500 U / L, and pH 7.2–7.4. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution I is 1.5–2.5% (w / v), and the concentration of DNase is 3600–4400 U / L. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution I is 1.8–2.2% (w / v), and the concentration of DNase is 3800–4200 U / L. In the most preferred embodiment, the concentration of sodium dodecyl sulfate in decellularization solution I is 2.0% (w / v), and the concentration of DNase is 4000 U / L. The components of decellularization solution II may be: sodium dodecyl sulfate at a concentration of 0.6–1.5% (w / v), DNase at a concentration of 2500–3500 U / L, and pH 7.2–7.4. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution II is 0.7–1.3% (w / v), and the concentration of DNase is 2600–3400 U / L. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution II is 0.8–1.2% (w / v), and the concentration of DNase is 2800–3200 U / L. In the most preferred embodiment, the concentration of sodium dodecyl sulfate in decellularization solution II is 1.0% (w / v), and the concentration of DNase is 3000 U / L.

[0014] The gradient can also be a gradient consisting of decellularization solution I, decellularization solution II, and decellularization solution III. For example, the components of decellularization solution I and decellularization solution II are the same as those of the aforementioned decellularization solution I and decellularization solution II, while the components of decellularization solution III can be: sodium dodecyl sulfate concentration of 0.1–0.6% (w / v), DNase concentration of 1500–2500 U / L, and pH 7.2–7.4. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution III is 0.2–0.6% (w / v), and the concentration of DNase is 1600–2400 U / L. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution III is 0.4–0.6% (w / v), and the concentration of DNase is 1800–2200 U / L. In the most preferred embodiment, the concentration of sodium dodecyl sulfate in decellularization solution III is 0.5% (w / v), and the concentration of DNase is 2000 U / L.

[0015] According to another aspect of the present invention, the use of the decellularized umbilical cord matrix obtained by the above method or the use of the decellularized umbilical cord matrix obtained by the above method is provided for use as a substrate for cell culture, tissue damage repair, medical aesthetics, and cosmetic procedures.

[0016] Some embodiments of the present invention offer the following advantages: In some embodiments, the preparation method involves using one umbilical vein and at least one umbilical artery, employing an optimized perfusion rate and a gradient decellularized solution to fill the umbilical cord and create a certain degree of osmotic pressure, thereby eluting the cells within the umbilical cord. Some embodiments utilize a programmed perfusion method, filling a gap in the preparation of decellularized umbilical cord matrix. While removing the original resident cells of the umbilical cord tissue, the decellularized umbilical cord matrix retains the original vascular structure and extracellular matrix of the umbilical cord, such as Wharton's colloid, type I collagen, type III collagen, elastin, and laminin. Furthermore, some embodiments of the present invention better preserve the original structural proteins and active factors of the umbilical cord extracellular matrix while removing immunogenicity, which is of great significance for the development of novel regenerative and repair materials. Attached Figure Description

[0017] Figures 1A to 1C This is a gross observation diagram of the decellularized umbilical cord matrix and intermediates of its main steps prepared by the programmed perfusion method based on the present invention.

[0018] Figures 1D to 1F This is a gross observation diagram of the decellularized umbilical cord matrix prepared based on the traditional oscillation method and the intermediates of its main steps.

[0019] Figure 2A , Figure 2B , Figure 2C These are histological staining images of the umbilical cord in its natural state, the decellularized umbilical cord matrix prepared by programmed perfusion, and the decellularized umbilical cord matrix prepared by shaking method.

[0020] Figure 3A , Figure 3B , Figure 3C , Figure 3D These are, respectively, an identification diagram and a quantitative analysis diagram of type I collagen in a naturally occurring umbilical cord, a decellularized umbilical cord matrix prepared by a programmed perfusion method, and a decellularized umbilical cord matrix prepared by a shaking method.

[0021] Figure 4A , Figure 4B , Figure 4C , Figure 4D These are, respectively, the identification diagram and quantitative analysis diagram of type III collagen in the natural state umbilical cord, the decellularized umbilical cord matrix prepared by the programmed perfusion method, and the decellularized umbilical cord matrix prepared by the shaking method.

[0022] Figure 5A , Figure 5B , Figure 5C , Figure 5D These are, respectively, elastin identification diagrams and quantitative analysis diagrams of umbilical cord in its natural state, umbilical cord decellularized matrix prepared by programmed perfusion method, and umbilical cord decellularized matrix prepared by shaking method.

[0023] Figure 6A , Figure 6B , Figure 6C , Figure 6D These are images of laminin identification and quantitative analysis of natural umbilical cord, decellularized umbilical cord matrix prepared by programmed perfusion method, and decellularized umbilical cord matrix prepared by shaking method. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] In the description of this invention, unless otherwise defined, the following units have the following meanings: g represents gram, μm represents micrometer, mm represents millimeter, L represents liter, mL represents milliliter, ℃ represents degree Celsius, min represents minute, h represents hour, U / L represents unit / liter, rpm represents revolutions per minute; μL / min represents microliter per minute; w / v represents weight-to-volume ratio.

[0026] The method for preparing decellularized umbilical cord matrix of the present invention is applicable to the preparation of umbilical cords from all organisms, such as those from non-human mammals, including pig, bovine, and sheep umbilical cords. The following detailed description will use a pig umbilical cord as an example. All specific parameters mentioned in the following embodiments are for illustrative purposes only. Those skilled in the art can derive various simple variations from the disclosure below, and these variations are also within the scope of protection of the present invention.

[0027] It should be understood that the umbilical cord may be a fresh, unfrozen umbilical cord, or a fresh umbilical cord that has been transported frozen and then thawed. The umbilical cord may be cleaned with phosphate buffer and / or saline solution. For example, a fresh porcine umbilical cord may be cleaned multiple times in phosphate buffer to remove umbilical cord blood and clots.

[0028] It should be understood that the umbilical cord can be the entire umbilical cord or a segment of the umbilical cord, such as half an umbilical cord, one-third of an umbilical cord, or a smaller or larger segment, as long as it contains the umbilical vein and umbilical artery used in the method of the present invention.

[0029] The umbilical cord decellularization fluid used, as well as the reagents and equipment required for identification, include:

[0030] Phosphate buffer: Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4, dissolve them in 1L of ultrapure water, adjust the pH to 7.2-7.4, autoclave at 121℃ for 20min, and store at 4℃.

[0031] Decellularized matrix digestion solution: Dissolve 0.1g of pepsin in 100mL of acetic acid solution, adjust the pH to 2.0, and filter through a 0.22μm microporous membrane for sterilization. Prepare fresh before use for the digestion of decellularized matrices.

[0032] Decellularization solutions: The provided decellularization solutions I, II, and III are used to form a concentration gradient for decellularizing the umbilical cord. The gradient can be a gradient formed by decellularization solutions I and II, or it can be a gradient formed by decellularization solutions I, II, and III.

[0033] The decellularization solution I may contain the following components: sodium dodecyl sulfate at a concentration of 1.5–3.5% (w / v), DNase at a concentration of 3500–4500 U / L, and pH 7.2–7.4. In some more preferred embodiments, the sodium dodecyl sulfate concentration in decellularization solution I is 1.5–2.5% (w / v), and the DNase concentration is 3600–4400 U / L. In some even more preferred embodiments, the sodium dodecyl sulfate concentration in decellularization solution I is 1.8–2.2% (w / v), and the DNase concentration is 3800–4200 U / L. In the most preferred embodiment, the sodium dodecyl sulfate concentration in decellularization solution I is 2.0% (w / v), and the DNase concentration is 4000 U / L.

[0034] The components of decellularization solution II may be: sodium dodecyl sulfate at a concentration of 0.6–1.5% (w / v), DNase at a concentration of 2500–3500 U / L, and pH 7.2–7.4. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution II is 0.7–1.3% (w / v), and the concentration of DNase is 2600–3400 U / L. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution II is 0.8–1.2% (w / v), and the concentration of DNase is 2800–3200 U / L. In the most preferred embodiment, the concentration of sodium dodecyl sulfate in decellularization solution II is 1.0% (w / v), and the concentration of DNase is 3000 U / L.

[0035] The components of decellularization solution III may be: sodium dodecyl sulfate at a concentration of 0.1–0.6% (w / v), DNase at a concentration of 1500–2500 U / L, and pH 7.2–7.4. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution III is 0.2–0.6% (w / v), and the concentration of DNase is 1600–2400 U / L. In some more preferred embodiments, the concentration of sodium dodecyl sulfate in decellularization solution III is 0.4–0.6% (w / v), and the concentration of DNase is 1800–2200 U / L. In the most preferred embodiment, the concentration of sodium dodecyl sulfate in decellularization solution III is 0.5% (w / v), and the concentration of DNase is 2000 U / L.

[0036] The choice of perfusion vessel is crucial for effective decellularization. The umbilical cord comprises the umbilical vein and two umbilical arteries. The inventors discovered that using only one umbilical vessel (either an umbilical artery or vein) as the perfusion pathway yields unsatisfactory decellularization results regardless of perfusion rate or filling depth. Using two umbilical vessels (two arteries or one vein plus one artery) provides better decellularization results, but two arteries alone do not achieve satisfactory results. Using the umbilical vein and one artery yields even better results. Using three umbilical vessels (an umbilical vein plus two arteries) achieves the best decellularization effect. Table 1 shows the cell retention results after perfusion via different umbilical vessels and the cell retention using the shaking method.

[0037] Table 1:

[0038]

[0039] in,

[0040] "*" indicates the gradient of cell nuclei per unit area, and the natural umbilical cord is defined as *****;

[0041] "——" indicates nothing;

[0042] Since there is no internal circulation between arteries and veins in the umbilical cord, the perfusion rate should be adjusted to fill the umbilical cord and form a certain degree of osmotic pressure, without overfilling the blood vessels or even causing them to rupture, so as to wash away the cells inside the umbilical cord.

[0043] The inventors discovered that different flow rates of decellularization fluid have varying effects. Because the umbilical cord itself has relatively low structural strength, excessively high flow rates of the decellularization fluid can over-impact the umbilical vessels and cord, potentially damaging the umbilical matrix and causing loss of Wharton's globulin. Conversely, excessively low flow rates fail to effectively remove cells. Therefore, in addition to selecting the appropriate decellularization fluid, it is necessary to control its flow rate to regulate the amount of fluid flowing through a unit area of ​​the umbilical vessels. The inventors found that for different perfusion pathways, a perfusion rate of 100 μL / min to 1500 μL / min is optimal.

[0044] In addition, the degree of filling during perfusion also affects the result of umbilical cord decellularization. For example, the umbilical cord needs to be filled but not overfilled, which could cause the blood vessels to rupture or even the umbilical cord itself. At the same time, it is also necessary to ensure that the cellular components damaged by the decellularization fluid are flushed out. Therefore, the diameter of the umbilical cord after filling should be 1.2 to 2.5 times the diameter of the umbilical cord before perfusion.

[0045] In some embodiments of the present invention, a specific programmed perfusion method is used for decellularization, for example, perfusing the cells for a first time period, a second time period, a third time period, and a fourth time period with the aforementioned decellularization solutions I, II, III, and phosphate buffer, respectively. The first, second, third, and fourth time periods can be the same or different time periods. For example, they can be any time period between 5 and 7 hours, such as 5 hours, 6 hours, 7 hours, etc. It should be understood that the above-mentioned limitations on the perfusion time periods are only preferred, and similar or longer perfusion times can also be used to achieve similar effects.

[0046] After perfusion using the above procedure, the umbilical cord can be fixed with 4% paraformaldehyde, prepared into slides, and stained using the hematoxylin-eosin staining method, i.e., HE staining. The stained slides are then subjected to panoramic scanning. By observing the preservation of cell nuclei and comparing the HE staining results with those of natural, undecellularized umbilical cord tissue and umbilical cord tissue obtained through shaking and elution, the extent of cell removal is reflected.

[0047] Based on the above exploration of umbilical cord decellularization methods and the optimal preservation of subsequent tissue integrity and extracellular matrix proteins, a gradient concentration decellularization method was further optimized to reduce the damage to the matrix during the decellularization process.

[0048] Example 1: Preparation of decellularized umbilical cord matrix using programmed perfusion method.

[0049] In this embodiment, freshly collected and frozen pig umbilical cords were thawed and used to install the infusion apparatus, with the following results: Figure 1AAs shown. The umbilical cord should be rinsed three times in phosphate-buffered saline (PBSS) to ensure the removal of blood and clots. For example, the pig umbilical cord can be perfused with PBSS through three umbilical vessels (two umbilical arteries and one umbilical vein) for 1 hour. The perfusion rate should be sufficient to create sufficient osmotic pressure to fill the umbilical cord without over-filling or rupturing the vessels. For example, a perfusion rate of 100 μL / min per vessel is recommended. The diameter of the filled umbilical cord should be 1.2 times the diameter before perfusion. The cleaned pig umbilical cord is shown below. Figure 1B As shown.

[0050] The umbilical cord was perfused for 6 hours using cell-exfoliating solution I (2.0% (w / v) sodium dodecyl sulfate solution, 4000 U / L DNase, pH 7.2); followed by cell-exfoliating solution II (1.0% (w / v) sodium dodecyl sulfate solution, 3000 U / L DNase, pH 7.2); and then cell-exfoliating solution III (0.5% (w / v) sodium dodecyl sulfate solution, 2000 U / L DNase, pH 7.2). The perfusion rate was 100 μL / min to fill the umbilical cord to 1.2 times its original diameter. Afterward, phosphate buffer was used for 6 hours to remove residual cell-exfoliating solution, yielding the decellularized umbilical cord matrix. Figure 1C As shown. Comparative Example 1: Preparation of decellularized umbilical cord matrix using conventional elution method.

[0051] Freshly collected and frozen pig umbilical cords were thawed, cut into small pieces, and washed three times with heparinized phosphate buffer. The results were as follows: Figure 1D As shown; the umbilical cord was washed with decellularized solution I for 6 hours by shaking; the umbilical cord was washed with decellularized solution II for 12 hours by shaking; the umbilical cord was washed with decellularized solution III for 6 hours by shaking; wherein decellularized solutions I, II, and III were the same as in Example 1. The results are as follows. Figure 1E As shown; the umbilical cord was washed with phosphate buffer for 6 hours to remove residual decellularized solution, resulting in the decellularized umbilical cord matrix as shown. Figure 1F As shown.

[0052] Histological examination of decellularized umbilical cord matrix

[0053] Natural umbilical cord tissue and decellularized umbilical cord matrix obtained by the two methods described above were fixed with 4% paraformaldehyde, prepared into slides, and stained with hematoxylin and eosin (HE). Panoramic scanning of the stained slides was performed to observe cell removal and structural changes, and comparative analysis was conducted. The results showed that, compared to… Figure 2A As shown in the natural umbilical cord tissue, decellularization can effectively remove cellular components from the umbilical cord, such as... Figure 2B and Figure 2C As shown, compared with the decellularized umbilical cord matrix obtained by the traditional oscillation method, the programmed perfusion method proposed in this invention preserves the original structure of the umbilical cord and Wharton's jelly components more completely.

[0054] Type I collagen detection in decellularized umbilical cord matrix

[0055] Umbilical cord tissue and decellularized umbilical cord matrix obtained by two different preparation methods were fixed with 4% paraformaldehyde, prepared into slides, and subjected to immunohistochemical staining for type I collagen. Panoramic scanning of the stained slides was used to observe the retention and structural changes of type I collagen, and data were acquired using ImageJ, with quantitative comparative analysis based on average optical density (AOD). The results showed that compared to… Figure 3A The umbilical cord in its natural state, and Figure 3C The oscillation method shown yields decellularized umbilical cord matrix, such as... Figure 3B The perfusion method of the embodiment of the present invention shown in the figure better preserves the histological structure of the umbilical cord; Figure 3D Analysis showed that the perfusion method preserved type I collagen better than the oscillation method (*p<0.05).

[0056] Type III collagen detection in decellularized umbilical cord matrix

[0057] Umbilical cord tissue and decellularized umbilical cord matrix obtained by two different preparation methods were fixed with 4% paraformaldehyde, prepared into slides, and subjected to immunohistochemical staining for type III collagen. Panoramic scanning of the stained slides was used to observe the retention and structural changes of type III collagen, and data were acquired using ImageJ, with quantitative comparative analysis based on average optical density (AOD). Results showed that compared to… Figure 4A The umbilical cord in its natural state, and Figure 4C The oscillation method shown yields decellularized umbilical cord matrix, such as... Figure 4B The perfusion method of the embodiment of the present invention shown in the figure better preserves the histological structure of the umbilical cord; Figure 4D Analysis showed that, compared with the oscillation method, the perfusion method has a better tendency to preserve type III collagen.

[0058] Elastin detection in decellularized umbilical cord matrix

[0059] Umbilical cord tissue and decellularized umbilical cord matrix obtained by two different preparation methods were fixed with 4% paraformaldehyde, prepared into slides, and subjected to immunohistochemical staining of elastin. Panoramic scanning of the stained slides was used to observe the retention and structural changes of elastin, and data were acquired using ImageJ, with quantitative comparative analysis based on average optical density (AOD). The results showed that compared to… Figure 5A The umbilical cord in its natural state, and Figure 5C The oscillation method shown yields decellularized umbilical cord matrix, such as... Figure 5B The perfusion method of the embodiment of the present invention shown in the figure better preserves the histological structure of the umbilical cord; Figure 5DAnalysis showed that the perfusion method preserved elastin better than the shaking method (*p<0.05). Laminin detection in decellularized umbilical cord matrix.

[0060] Umbilical cord tissue and decellularized umbilical cord matrix obtained by two different preparation methods were fixed with 4% paraformaldehyde, prepared into slides, and subjected to immunohistochemical staining for laminin. Panoramic scanning of the stained slides was used to observe the retention and structural changes of laminin, and data were acquired using ImageJ, with quantitative comparative analysis based on average optical density (AOD). The results showed that compared to… Figure 6A The umbilical cord in its natural state, and Figure 6C The oscillation method shown yields decellularized umbilical cord matrix, such as... Figure 6B The perfusion method of the embodiment of the present invention shown in the figure better preserves the histological structure of the umbilical cord; Figure 6D Analysis showed that the perfusion method preserved laminin better than the oscillation method (**p<0.01).

[0061] Example 2:

[0062] Using fresh porcine umbilical cord as the sample, after washing, the cord was perfused with decellularized solution I for 5 hours; then with decellularized solution II for 5 hours; and finally with decellularized solution III for 5 hours. The perfusion rate was 1500 μL / min to fill the umbilical cord to approximately 2.5 times its original diameter before perfusion. This prevented over-inflation of blood vessels or even rupture. The cord was then perfused with phosphate buffer for 5 hours to remove residual decellularized solution, yielding the decellularized umbilical cord matrix. The decellularized solutions I, II, and III were the same as in Example 1.

[0063] Using the same fixation, staining, and scanning methods as in Example 1, it can be seen that the original structure of the umbilical cord and the Wharton's colloid components are basically intact and better preserved than the shaking and washing method. Elastin, laminin, type I collagen, and type III collagen in the umbilical cord are also better preserved.

[0064] Example 3:

[0065] Using fresh porcine umbilical cord as the sample, after washing, the cord was perfused with decellularized solution I for 7 hours; then with decellularized solution II for 7 hours; and finally with decellularized solution III for 7 hours. The perfusion rate was 500 μL / min to fill the umbilical cord to approximately twice its original diameter before perfusion. This prevented over-inflation of blood vessels or even rupture. The cord was then perfused with phosphate buffer for 7 hours to remove residual decellularized solution, yielding the decellularized umbilical cord matrix. The decellularized solutions I, II, and III were the same as in Example 1.

[0066] Using the same fixation, staining, and scanning as in Example 1, it can be seen that the original structure of the umbilical cord and the Wharton's colloid components are basically intact and better preserved than the shaking and washing method. Elastin, laminin, type I collagen, and type III collagen in the umbilical cord are also better preserved.

[0067] Disinfection can be performed at any step of the method of the present invention, such as pre-sterilization of the entire perfusion system using the disinfection techniques involved, perfusion with sterile liquid throughout the process, perfusion with disinfectant solution after preparation, or radiation sterilization. The prepared decellularized umbilical cord matrix can be preserved by hydration or by vacuum freeze-drying to form a dried body.

[0068] The decellularized umbilical cord matrix prepared by this invention can be used to derive medical products such as microparticles, fluidized compositions, gels, and active peptides. The microparticle products are obtained by vacuum freeze-drying the decellularized umbilical cord matrix followed by high-speed rotary pulverization, and include multiple product grades (≤38μm, 38–100μm, 100–250μm), which can be used as wound coverings and filling materials for cell defects. The fluidized compositions are obtained by digesting the decellularized umbilical cord matrix microparticles with pepsin in an acidic environment. The gel products are obtained by further concentrating and neutralizing the homogeneous fluidized compositions, and include different concentration grades, which can be used for wound coverings and injectable filling of skin defects. The techniques for preparing the above-mentioned derivative products are well known to those skilled in the art.

[0069] The applications of the decellularized umbilical cord matrix and its derivative medical products prepared by this invention include in vitro construction in the fields of tissue engineering and regenerative medicine, and repair of various types of tissue defects.

[0070] Traditional decellularization methods mainly include vascular perfusion and tissue block elution. Both methods are suitable for common tissues such as the heart and liver. However, for the umbilical cord, the inventors discovered that the Wharton's colloid in the umbilical cord tissue has low toughness. Tissue block elution causes the colloid to loosen and leak, resulting in the loss of many components, as shown in the attached figures. While perfusion has a significant advantage in preserving active substances, the umbilical cord differs from conventional organs, which can be perfused throughout the entire organ via arteriovenous and capillary networks. The umbilical cord is a "severed" tissue, lacking closed loops between arteries and veins and capillaries. Furthermore, the inventors found that umbilical cord vessels are also excellent materials, promoting angiogenesis and wound healing; therefore, they should be preserved as much as possible during processing and not removed.

[0071] Therefore, this invention analyzed decellularization methods using different blood vessels and under certain osmotic pressures, and finally explored the most effective umbilical cord decellularization method proposed in this invention, which thoroughly decellularizes while completely preserving the structure and extracellular matrix components of the umbilical cord.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the substantive technical content of the present invention. The substantive technical content of the present invention is broadly defined within the scope of the claims. Any technical entity or method completed by others that is completely identical to or an equivalent modification of the claims is considered to be covered within the scope of the claims.

Claims

1.A method for preparing an umbilical cord acellular matrix based on a perfusion elution procedure, comprising the following steps: a.pretreating an umbilical cord; b.perfusing physiological saline and / or phosphate buffer through blood vessels of the umbilical cord to remove blood in the umbilical cord; c.perfusing an acellular solution through blood vessels of the umbilical cord to elute cells in the umbilical cord; d.perfusing a phosphate buffer through blood vessels of the umbilical cord to remove residual acellular solution in the umbilical cord to obtain the umbilical cord acellular matrix; wherein: in step c, one umbilical vein and one umbilical artery, or one umbilical vein and two umbilical arteries are used as perfusion paths; and acellular solution I, acellular solution II, and acellular solution III are used for perfusion in sequence; wherein acellular solution I comprises 1.5-3.5% w / v sodium dodecyl sulfate, 3500-4500 U / L DNase, and pH 7.2-7.4; acellular solution II comprises 0.6-1.5% w / v sodium dodecyl sulfate, 2500-3500 U / L DNase, and pH 7.2-7.4; and acellular solution III comprises 0.1-0.6% w / v sodium dodecyl sulfate, 1500-2500 U / L DNase, and pH 7.2-7.4; the perfusion speed of the acellular solution is 100-1500 μL / min; the perfusion time of the acellular solution is 5-7 hours; and the diameter of the perfused umbilical cord is 1.2-2.5 times the diameter of the unperfused umbilical cord. The method further comprises cutting, lyophilizing, and grinding the prepared umbilical cord acellular matrix to obtain umbilical cord acellular matrix lyophilized powder. The method further comprises weighing the umbilical cord acellular matrix lyophilized powder, stirring and enzymatic hydrolysis in a protease solution, adjusting the pH value, and obtaining a solution. The method further comprises lyophilizing the solution again to obtain secondary lyophilized powder of the umbilical cord acellular matrix. The umbilical cord is derived from a non-human mammal. 5.An umbilical cord acellular matrix obtained by the method of any one of claims 1 to 4. The umbilical cord acellular matrix is used for preparing a reagent for repairing soft tissue injury, medical cosmetology, or life cosmetology. ​ 2. The method of claim 1, wherein: ​ 3. The method of claim 2, wherein the umbilical cord decellularized matrix is prepared by the steps of: ​ ​ 4. The method of claim 1, wherein the umbilical cord decellularized matrix is prepared by the steps of: ​ ​ 6. Use of the umbilical cord decellularized matrix as claimed in claim 5, characterized in that, ​

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