Tissue engineering skin with skin appendage reconstruction function and preparation method and application thereof

Abstract

The invention discloses tissue engineering skin with a skin appendage reconstruction function and a preparation method and application thereof. The tissue engineering skin with the skin appendage reconstruction function is obtained by taking a decellularized pigskin matrix containing a basement membrane band as a scaffold material and planting a hiPSCs-sourced skin-imitating cell component seed on the scaffold material and then culturing the hiPSCs-sourced skin-imitating cell component seed. The stent material is a decellularized pigskin matrix containing a basement membrane band, and the basement membrane band is of a structure lacked by stent materials in the market at present. However, the basilar membrane has the functions of supporting and connecting the epidermal layer and the corium layer, signal transduction and permeation barrier, so that the basilar membrane is very important in tissue engineering skin. Meanwhile, the decellularized pigskin scaffold is good in biocompatibility, high in biological activity and good in strength and toughness, and materials are easy to obtain.

Description

Technical Field

[0001] The present invention belongs to the fields of tissue engineering, materials science and biology, and specifically relates to a tissue-engineered skin with skin appendage reconstruction function, a preparation method and an application thereof. Background Art

[0002] Scaffold materials are artificial. When used clinically, cells on artificial scaffolds tend to grow unevenly and infiltrate poorly, resulting in significantly prolonged wound healing time with increasing scaffold thickness. Artificial scaffolds may present biocompatibility issues, with evidence suggesting they may induce inflammatory responses and hinder wound re-epithelialization in humans. Artificial scaffolds lack active cells and cytokine modification. Even if artificially added, their short half-life in the human body limits their clinical application. For patients with severe skin damage and complete loss of nerves to the skin appendages (hair follicles, sweat glands, sebaceous glands) in the donor site, the use of artificial scaffold materials alone will not provide a repairing effect.

[0003] Currently, seed cells come from sources such as the patient's own autologous cells, allogeneic cells, and xenogeneic cells. The main disadvantage of autologous cells is that they are very limited in availability, especially when the tissue is severely damaged or the body is in poor condition, and obtaining cells in good condition is not guaranteed. Elderly autologous cells have poor proliferation capacity and low ability to form new tissues. Harvesting the material will cause further damage to the body, so autologous cells cannot be further used in tissue engineering. The main disadvantage of allogeneic seed cells is that they are antigenic, requiring further research on antigenicity and further measures to reduce antigenicity. Cells are prone to aging, which leads to the loss of the ability to proliferate and secrete matrix. There is also the disadvantage of a large amount of material collected but a small amount of cell culture. Summary of the Invention

[0004] The purpose of the present invention is to provide a decellularized porcine skin matrix containing a basement membrane zone and a preparation method and application thereof.

[0005] Another object of the present invention is to provide a tissue-engineered skin with skin appendage reconstruction function, and a preparation method and application thereof.

[0006] To achieve the purpose of the present invention, in a first aspect, the present invention provides a method for preparing a cell-free pigskin matrix containing a basement membrane zone, comprising: depilating fresh pigskin and cutting it into small pieces, washing it with a PBS buffer solution, and then sequentially subjecting it to a decellularization treatment, a high-salt treatment, and a nuclease treatment, and finally rinsing it with a PBS buffer solution. The pigskin after the above treatments is freeze-dried and sterilized by irradiation to obtain the obtained matrix. Decellularization was performed using a 1% SDC (sodium deoxycholate) decellularization solution containing 2000 U / L PLA2 (phospholipase A2).

[0007] Preferably, hair is removed using waxing.

[0008] Furthermore, a 3-4 M (preferably 3.4 M) solution is used for high salt treatment.

[0009] Preferably, the nuclease treatment is performed using a solution containing 8-12 μg / ml DNase and 4-6 μg / ml RNase (preferably, the nuclease treatment is performed using a solution containing 10 μg / ml DNase and 5 μg / ml RNase).

[0010] In the present invention, the accession numbers of DNase and RNase in NCBI are P04406 and P00698 respectively.

[0011] Furthermore, the method comprises the following steps: 1) Take fresh, depilated pig skin and cut skin slices with a thickness of 100-750 μm on an ice table; 2) After washing with 4℃ pre-cooled PBS buffer, cut the skin into 10cm pieces 2 After sizing, the cells were placed in a 1% SDC decellularization solution containing 2000 U / L PLA2 and decellularized in a 37°C constant temperature shaker at 200-500 rpm (preferably 350 rpm); 3) Replace the decellularization solution every 1.5-2.5 hours (preferably 2 hours) until the porcine skin tissue turns milky white. Prepare quick frozen sections and stain with DAPI (300 nM) to ensure the absence of cell structures. 4) The cell-free skin sheet was washed with 4°C pre-cooled PBS buffer until no foam was present. The skin sheet was then placed in a 3-4M (preferably 3.4M) NaCl solution and shaken at room temperature for 1 hour. 5) Remove the high-salt-treated skin piece, rinse with PBS at room temperature, and place in a solution containing 8-12 μg / ml DNase and 4-6 μg / ml RNase (preferably 10 μg / ml DNase and 5 μg / ml RNase) at 37°C on a constant-temperature shaker at 200-500 rpm (preferably 350 rpm) for 1 hour. 6) Wash the skin slices after the above treatment with PBS buffer three times, each time for 10 minutes; 7) The cleaned skin pieces are freeze-dried and sterilized by cobalt-60 ionizing radiation.

[0012] In the present invention, the pigskin is from Bama pigs or Landrace pigs, preferably 50-80 days old Bama pigs.

[0013] In a second aspect, the present invention provides a decellularized porcine skin matrix containing a basement membrane zone prepared according to the method.

[0014] In a third aspect, the present invention provides a method for preparing tissue-engineered skin with skin appendage reconstruction function, using the decellularized porcine skin matrix containing the basement membrane zone as a scaffold material, and seeding hiPSCs-derived skin-mimicking cell components on the scaffold material, placing them in OMM culture medium, and culturing them at 37°C for 5-100 days.

[0015] The OMM culture medium is prepared by mixing Advanced DMEM-F12 culture medium (purchased from Gibco, product number 12634028), Neurobasal culture medium (purchased from Gibco, product number 21103049), B27 (purchased from Thermo Fisher Scientific, product number 17504044), Glutamax (purchased from Gibco, product number 35050), N2 (purchased from Gibco, product number 17502048), 2-mercaptoethanol, and Normocin, wherein the volume ratio of Advanced DMEM-F12 culture medium to Neurobasal culture medium is 1:1, so that the final concentrations of B27, Glutamax, N2, 2-mercaptoethanol, and Normocin (purchased from InvivoGen, product number Ant-nr-1) are (0.4-0.6)×, 1×, (0.4-0.6)×, and 0.09-0.11, respectively. mM, 90-110µg / mL (preferably, the final concentrations of B27, Glutamax, N2, 2-mercaptoethanol, and Normocin are 0.5×, 1×, 0.5×, 0.1mM, and 100µg / mL, respectively).

[0016] The preparation method of the skin-mimicking cell component seeds comprises: (1) Culture iPSCs in the mTeSR culture system to a cell density of 70%; (2) On day (-2), iPSCs were dissociated into single cells and aggregated in a 96-well plate at a concentration of 3000 viable cells / well to form embryoid bodies; In the present invention, the induction start time is set as day (-2); (3) On day 0 of differentiation, transfer the embryoid bodies to a new 96-well plate and add differentiation medium to induce ectoderm formation; culture conditions: 37°C, 5% CO2; The differentiation medium is: Essential 6 medium (purchased from Thermo Fisher, catalog number A1516401) supplemented with 1.5-2.5% Matrigel (purchased from Corning, catalog number 354230), 7.5-12.5 μM SB-431542 (purchased from Selleck, catalog number S1067), 3-5 ng / mL bFGF (purchased from Peprotech, catalog number 100-18B) and 2-3 ng / mL BMP4 (purchased from Peprotech, catalog number 120-05). Preferably, the differentiation medium is: Essential 6 medium supplemented with 2% Matrigel, 10 μM SB-431542, 4 ng / mL bFGF and 2.5 ng / mL BMP4. (4) On the third day of differentiation, 25 μl of E6LF medium was added to induce the formation of non-epithelial mesenchymal cells; culture conditions: 37°C, 5% CO2; The E6LF medium is: Essential 6 medium supplemented with 200-300 ng / mL bFGF and 0.75-1.25 μM LDN (purchased from Selleck, catalog number S7507). Preferably, the E6LF medium is: Essential 6 medium supplemented with 250 ng / mL bFGF and 1 μM LDN; (5) On the 6th day of differentiation, add 75 μl of Essential 6 medium to each well; on the 8th and 10th days, replace half of the medium with fresh Essential 6 medium; culture conditions: 37°C, 5% CO2; (6) On day 12 of differentiation, transfer the embryoid bodies to a 24-well plate, add 500 μl of OMM medium containing 1.5-2.5% (preferably 2%) Matrigel, and continue culturing on a shaker at 60-80 rpm (preferably 70 rpm) to induce further development of the skin cell component seeds. Culture conditions: 37°C, 5% CO2; (7) On day 15 of differentiation, replace half of the culture medium with fresh OMM medium containing 1% Matrigel. Starting from day 18, replace half of the culture medium with fresh OMM medium every 2-3 days until the skin-like cell components are mature. Culture conditions: 37°C, 5% CO2, place the well plate containing the skin organoids on a shaker at 60-80 rpm (preferably 70 rpm).

[0017] In the present invention, the chemical name of SB-431542 is: N-[2-(4-(2,4-dimethylphenyl)-thiazol-2-yl)thiazol-4-yl]-N'-hydroxyurea.

[0018] The chemical name of LDN (LDN-193189) is N-(3-(4-(Dimethylamino)phenyl)-5-methylisoxazol-4-yl)-N'-(2-hydroxyethyl)urea.

[0019] The mature skin-mimicking cell component seeds are planted on the scaffold material after 3D culture, and then flipped upside down and placed in OMM culture medium for culture; The scaffold material is pre-divided into small squares of 2 cm×2 cm using a sterile water pen to ensure that each small square contains one cell.

[0020] The 3D culture conditions are as follows: incubator temperature 37°C, CO2 concentration 5%, starting from day 12, the well plate containing the skin organoids was placed on a shaker with a shaker speed of 70 rpm.

[0021] In a fourth aspect, the present invention provides tissue-engineered skin with skin appendage reconstruction function prepared according to the method.

[0022] In a fifth aspect, the present invention provides applications of the acellular porcine skin matrix containing a basement membrane zone or the tissue-engineered skin with skin appendage reconstruction function in the field of skin tissue engineering.

[0023] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: In terms of scaffold material, the present invention uses decellularized pig skin that retains the basement membrane zone as the scaffold material: (1) Few tissue-engineered skin scaffolds on the market utilize pig skin as a scaffold material. A small number of pig skin dermal matrix dressings lack a basement membrane band, a structure currently lacking in scaffold materials on the market. However, the basement membrane supports and connects the epidermis to the dermis, conducts signals, and acts as a permeability barrier, making it crucial in tissue-engineered skin.

[0024] (2) The decellularized pigskin scaffold has good biocompatibility. The decellularized pigskin has been specially treated to remove cellular components, which reduces the occurrence of immune response and the risk of rejection.

[0025] (3) High biological activity: The product retains the structure and bioactive components of pig skin, which can promote the repair and regeneration of human tissues and accelerate the wound healing process.

[0026] (IV) Good strength and toughness: Decellularized pig skin for passive implantable devices has high strength and toughness, can provide good support and protection, and contribute to wound healing and tissue reconstruction.

[0027] (5) The quantity and area are sufficient because it is derived from pig skin, and the mature decellularization technology enables the decellularized pig skin scaffold with basement membrane band to be mass-produced.

[0028] In terms of seed cells, the present invention uses skin-like cell component seeds derived from human induced pluripotent stem cells (hiPSCs) to replace the single or several combined seed cell types transplanted in traditional tissue-engineered skin: (6) The hiPSCs-derived skin-mimicking cell component seeds cultured in the present invention contain keratinocytes, epidermal stem cells, hair follicles, sweat glands, sebaceous glands, and sensory neurons. Therefore, the hiPSCs-derived skin-mimicking cell component seeds are implanted on a decellularized pig skin scaffold containing a basement membrane zone, so that the tissue-engineered skin has cell types and cell ratios that are more similar to normal human skin tissue.

[0029] (VII) Using hiPSCs-derived skin-mimicking cell component seeds to lay on a decellularized pigskin scaffold with a basement membrane band can reasonably control the number of skin cells so that the number and composition of cells on the scaffold are uniform. Because each skin-mimicking cell component seed is visible to the naked eye, the decellularized pigskin scaffold with a basement membrane band can be artificially divided into small square blocks according to the area, and one skin-mimicking cell component seed is transplanted into each small square for co-culture.

[0030] (8) The skin-mimicking cell component seeds cultured in this invention are derived from hiPSCs and have been proven to be non-immunogenic and non-rejective through in vitro and in vivo experiments. They can be safely used in patients.

[0031] (IX) Non-invasive, no skin needs to be taken from the patient. The tissue-engineered skin cells in the present invention are derived from induced pluripotent stem cells. Therefore, the cells seeded with the skin cell components can be expanded indefinitely, and the cell number is sufficient to support patients with severe skin defects.

[0032] (10) The seeds of the simulated skin cell components contain a large number of epidermal stem / progenitor cells, so after being transplanted onto the decellularized pig skin scaffold, they can proliferate to a certain extent, allowing for uniform skin cell infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention provides a process flow for preparing acellular porcine skin matrix containing basement membrane bands in a preferred embodiment of the present invention.

[0034] Figure 2 DAPI staining of pig skin tissue before and after decellularization in a preferred embodiment of the present invention (scale bar 100µm).

[0035] Figure 3This is a preferred embodiment of the present invention showing continuous HE staining and immunohistochemical staining of 300µm thick PLA2-SDC decellularized pig skin (scale bar 50µm). COL4A1 (type IV collagen α1) and Laminin (laminin) are basement membrane zone-specific markers.

[0036] Figure 4 The following is a diagram showing the construction and verification of the simulated skin cell component seeds in a preferred embodiment of the present invention. Figure a is a flow chart for the culture of the skin cell component seeds in Example 2, which is a schematic diagram showing the key time points for the culture of the skin cell component seeds. Correspondingly, the bright field photographs on the right show the microscopic morphology of the skin cell component seeds at 16, 35, 55, 75, and 90 days (scale bar: 400 μm). Figure b is an immunofluorescence staining image of the skin cell component seeds at different time points (15 days, 35 days, 55 days, and 75 days) in Example 1. From day 15 to 35, the skin cell component seeds express epithelial cell markers (TFAP2A, ECAD, and KRT17). Starting from day 55, the skin cell component seeds express mesenchymal-related markers ( PDGFRα ), glial cell-related markers (SOX10 and P75), proliferation markers (PCNA), epidermal stem cell markers (KRT15 and KRT5), and starting from day 75, the skin cell component seeds expressed epidermal germ cell markers (LHX2, PCAD and EDAR) and dermal layer condensates (PDGFR α c is a bright field and immunofluorescence image of hair follicles (DAPI: cell nuclei) from the skin cell fraction seeded on day 90 described in Example 1 (scale bars: 200 and 50 µm). d is an immunofluorescence image of epithelial cell markers (ECAD), epidermal basal cell markers (KRT15, ​​KRT5, KRT14, and P63), mature epithelial markers (KRT1 and KRT10), proliferation marker (PCNA), desmosome (DSG1), and adhesion receptor (ITGB1) from the mature skin cell fraction seeded on day 120 described in Example 1 (scale bars: 200 and 50 µm). DETAILED DESCRIPTION

[0037] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0038] Example 1 Preparation of acellular porcine skin matrix containing basement membrane strips Operation process see Figure 1 .

[0039] 1. After anesthetizing 50-80 day old Bama pigs, quickly remove their hair with depilatory wax, remove the whole layer of fresh pig skin tissue, place it on an ice table, and adjust the thickness of the electric dermatome (100-750 μm) to remove the skin.

[0040] 2. After washing with 4℃ pre-cooled PBS buffer, cut the pig skin tissue into 10cm 2 After sizing, the cells were placed in 1% SDC decellularization solution containing 2000U / LPLA2 and decellularized in a constant temperature shaker at 37°C and 350rpm.

[0041] 3. Replace the decellularization solution every 2 hours until the pig skin tissue turns milky white. Quickly stain the frozen sections with DAPI (using a concentration of 300 nM) and incubate at room temperature for 10 minutes in the dark to observe the presence of cell nuclei to ensure that all cells are destroyed.

[0042] 4. After observing the cell-free structure, wash with 4°C pre-cooled PBS buffer until there is no foam. Oscillate with 3.4 M NaCl at room temperature for 1 hour. Check whether the optical density (OD) of the washing solution at 280 nm is negative for protein.

[0043] 5. After rinsing with PBS buffer at room temperature, place the sample in a solution containing DNase (10 μg / ml) and RNase (5 μg / ml) and incubate in a 37°C constant temperature shaker at 350 rpm for 1 hour. Check whether the optical density (OD) of the washing solution at 260 nm is negative for nucleic acid.

[0044] 6. Wash with PBS buffer three times, 10 minutes each time.

[0045] 7. Freeze-dry the decellularized pig skin and sterilize it with cobalt-60 ionizing radiation.

[0046] Example 2 Preparation of Skin-like Cell Component Seeds (1) iPSCs were cultured in the mTeSR culture system (mTeSR™1 Complete Kit, product number #85850, purchased from STEMCELL) to a cell density of 70%. iPSCs were obtained from the Medical Research Center of Peking Union Medical College Hospital.

[0047] (2) On day (-2), iPSCs were dissociated into single cells and aggregated in a 96-well plate at a concentration of 3000 viable cells / well to form embryoid bodies.

[0048] (3) On day 0 of differentiation, transfer the embryoid bodies to a new 96-well plate and add differentiation medium (Essential 6 medium supplemented with 2% Matrigel, 10 μM SB-431542, 4 ng / mL bFGF, and 2.5 ng / mL BMP4) to induce ectoderm formation. Incubate at 37°C with 5% CO2.

[0049] (4) On day 3 of differentiation, add 25 μl of E6LF medium (Essential 6 medium supplemented with 250 ng / mL bFGF and 1 μM LDN) to induce non-epithelial mesenchymal cell formation. Incubator temperature: 37°C, CO2 concentration: 5%.

[0050] (5) On day 6, add 75 μl of Essential 6 Medium (Cat. No. A1516401, Thermo Fisher Scientific) to each well. On days 8 and 10, replace half of the medium with fresh Essential 6 Medium. Incubator temperature: 37°C, CO2 concentration: 5%.

[0051] (6) On day 12, the embryoid bodies were transferred to a 24-well plate and 500 μl of OMM medium (50 mL containing 24.5 ml Advanced DMEM-F12 medium, 24.5 ml Neurobasal medium, 500 μl B27, 500 μl Glutamax, 250 μl N2, 91 μl 2-mercaptoethanol, and 100 μl Normocin) supplemented with 2% Matrigel was added. The plates were then placed on a shaker at 70 rpm to induce further development of the skin-like cell component seeds. The incubator temperature was 37°C, the CO2 concentration was 5%, and the well plates containing the skin organoids were placed on a shaker at 70 rpm.

[0052] (7) On day 15, replace half of the medium with fresh OMM medium supplemented with 1% Matrigel. Starting from day 18, replace half of the medium with fresh OMM medium every 2-3 days until the skin-like cell components are mature. The incubator temperature is 37°C, the CO2 concentration is 5%, and the well plate containing the skin organoids is placed on a shaker at 70 rpm.

[0053] Example 3 In vitro recellularization of acellular porcine skin matrix (1) Freeze-dried and ionizing radiation sterilized decellularized porcine skin matrix with basement membrane bands was used as the scaffold material (the decellularized porcine skin matrix was divided into 2 cm × 2 cm small squares with a sterile pen in advance.

[0054] (2) The tail of the 3D cultured skin-like cell component was removed and inoculated onto the basement membrane side of the decellularized pig skin, ensuring that each small square contained one skin-like cell component.

[0055] The 3D culture conditions were: incubator temperature 37°C, CO2 concentration 5%, and culture medium OMM.

[0056] (3) Turn the constructed tissue-engineered skin over and add fresh OMM culture medium for 5-100 days. The incubator temperature is 37°C and the CO2 concentration is 5%. The culture medium is OMM, and the medium is changed every two days, half of the volume. Initially, add 5 ml of OMM to a 10 cm dish each time, then aspirate and discard 2.5 ml each time and add 2.5 ml of fresh OMM. Finally, a tissue-engineered full-thickness skin containing skin appendages, sensory nerves, and epidermal stem cells is constructed.

[0057] like Figure 2 As shown, the decellularization method used in the present invention can remove all pig skin cells, thereby reducing immunogenicity.

[0058] like Figure 3 As shown in the figure, the dermal structure of the acellular pigskin is intact, similar to that of normal pigskin, and the texture of the basement membrane after removing the epidermis can be clearly seen.

[0059] The retained collagen fibers and other extracellular matrix components with low immunogenicity are abundant, which is beneficial to cell growth and proliferation.

[0060] The construction and staining verification of the skin-like cell component seeds can be found in Figure 4 .

[0061] Compared with tissue-engineered skin on the market, the present invention has the following main advantages: First, it is loaded with seeds of simulated skin cell components, which can reconstruct skin cells and appendages, such as keratinocytes, epidermal stem cells, hair follicles, sweat glands, sebaceous glands, sensory neurons, etc. Moreover, the seeds of simulated skin cell components come from hiPSCs, and their cell types and numbers are closer to those in real human skin epidermal cells, and they are non-immunogenic and will not produce rejection reactions. The scaffold material of the present invention is a decellularized pig skin matrix containing a basement membrane band, and the basement membrane band is a structure that is currently lacking in scaffold materials on the market. However, the basement membrane has the function of supporting and connecting the epidermis and dermis, the function of signal conduction, and the function of permeability barrier, so it is very important in tissue-engineered skin. At the same time, the decellularized pig skin scaffold has good biocompatibility, high biological activity, good strength and toughness, and is easy to obtain.

[0062] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing acellular porcine skin matrix containing a basement membrane zone, characterized in that: Fresh pig skin is dehaired and cut into small pieces, washed with PBS buffer, then subjected to decellularization treatment, high salt treatment, nuclease treatment, and finally rinsed with PBS buffer. The pig skin after the above treatment is freeze-dried and irradiated for sterilization. Among them, 1% SDC decellularization solution containing 2000U / L PLA2 was used for decellularization treatment.

2. The method according to claim 1, characterized in that Use waxing to remove hair; High salt treatment was performed using 3-4 M NaCl solution; Nuclease treatment was performed using a solution containing 8-12 μg / ml DNase and 4-6 μg / ml RNase.

3. The method according to claim 2, characterized in that The following steps are involved: 1) Take fresh, depilated pig skin and cut skin slices with a thickness of 100-750 μm on an ice table; 2) After washing with 4℃ pre-cooled PBS buffer, cut the skin into 10cm pieces 2 After sizing, the cells were placed in 1% SDC decellularization solution containing 2000 U / L PLA2 and decellularized in a constant temperature shaker at 37°C at 200-500 rpm; 3) Replace the decellularization solution every 1.5-2.5 hours until the porcine skin tissue turns milky white. Prepare quick frozen sections and stain with DAPI to ensure the absence of cell structures. 4) Wash the cell-free skin sheet with 4°C pre-cooled PBS buffer until no foam is left. Then, place the skin sheet in 3-4M NaCl solution and shake at room temperature for 1 hour. 5) Remove the high-salt-treated skin piece, rinse with PBS buffer at room temperature, and place in a solution containing 8-12 μg / ml DNase and 4-6 μg / ml RNase at 37°C on a constant temperature shaker at 200-500 rpm for 1 hour. 6) Wash the skin slices after the above treatment with PBS buffer three times, each time for 10 minutes; 7) The cleaned skin pieces are freeze-dried and sterilized by cobalt-60 ionizing radiation.

4. The method according to claim 3, characterized in that The pigskin is from Bama pigs or Landrace pigs, preferably 50-80 days old Bama pigs.

5. A cell-free porcine skin matrix containing a basement membrane zone prepared according to the method of any one of claims 1 to 4.

6. A method for preparing tissue-engineered skin with skin appendage reconstruction function, characterized in that: The acellular porcine skin matrix containing the basement membrane zone according to claim 5 is used as a scaffold material, hiPSCs-derived skin-mimicking cell component seeds are seeded on the scaffold material, placed in OMM culture medium, and cultured at 37° C. for 5-100 days; The OMM culture medium is prepared by mixing Advanced DMEM-F12 culture medium, Neurobasal culture medium, B27, Glutamax, N2, 2-mercaptoethanol, and Normocin, wherein the volume ratio of Advanced DMEM-F12 culture medium to Neurobasal culture medium is 1:1, so that the final concentrations of B27, Glutamax, N2, 2-mercaptoethanol, and Normocin are (0.4-0.6)×, 1×, (0.4-0.6)×, 0.09-0.11 mM, and 90-110 μg / mL, respectively.

7. The method according to claim 6, characterized in that The preparation method of the simulated skin cell component seeds comprises: (1) Culture iPSCs in the mTeSR culture system to a cell density of 70%-80%; (2) On day (-2), iPSCs were dissociated into single cells and aggregated in a 96-well plate at a concentration of 3000 viable cells / well to form embryoid bodies; The start time of induction was set as day (-2); (3) On day 0 of differentiation, transfer the embryoid bodies to a new 96-well plate and add differentiation medium to induce ectoderm formation; culture conditions: 37°C, 5% CO2; The differentiation medium is: Essential 6 medium supplemented with 1.5-2.5% Matrigel, 7.5-12.5 μM SB-431542, 3-5 ng / mL bFGF, and 2-3 ng / mL BMP4; (4) On the third day of differentiation, 25 μl of E6LF medium was added to induce the formation of non-epithelial mesenchymal cells; culture conditions: 37°C, 5% CO2; The E6LF medium is: Essential 6 medium supplemented with 200-300 ng / mL bFGF and 0.75-1.25 μM LDN; (5) On the 6th day of differentiation, add 75 μl of Essential 6 medium to each well; on the 8th and 10th days, replace half of the medium with fresh Essential 6 medium; culture conditions: 37°C, 5% CO2; (6) On day 12 of differentiation, transfer the embryoid bodies to a 24-well plate, add 500 μl of OMM medium containing 1.5-2.5% Matrigel, and continue culturing on a shaker at 60-80 rpm to induce further development of the skin-like cell component seeds; culture conditions: 37°C, 5% CO2; (7) On the 15th day of differentiation, replace half of the medium with fresh OMM medium containing 1% Matrigel; starting from the 18th day, replace half of the medium with fresh OMM medium every 2-3 days until the skin-like cell component seeds mature; culture conditions: 37°C, 5% CO2, shaker speed 60-80 rpm.

8. The method according to claim 7, characterized in that The mature skin-mimicking cell component seeds are planted on the scaffold material after 3D culture, and then flipped upside down and placed in OMM culture medium for culture; The scaffold material is pre-divided into 2 cm × 2 cm small squares using a sterile pen, ensuring that each small square contains one cell; The 3D culture conditions are as follows: incubator temperature of 37° C., CO 2 concentration of 5%, and culture on a shaker at a rotation speed of 60-80 rpm starting from day 12.

9. Tissue-engineered skin with skin appendage reconstruction function prepared according to the method according to any one of claims 6 to 8.

10. Use of the acellular porcine skin matrix containing a basement membrane zone according to claim 5 or the tissue-engineered skin with skin appendage reconstruction function according to claim 9 in the field of skin tissue engineering.

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