Ready-to-use skin seed cell as well as preparation method and application thereof

Abstract

The invention discloses a ready-to-use skin seed cell and a preparation method and application thereof.The ready-to-use skin seed cell takes hPSC cells as a source of seed cells and comprises hPSC-KC and / or hPSC-FB differentiated from the hPSC cells, and the preparation method comprises the steps that the hPSC cells are cultured; the hPSC cells are differentiated into hPSC-KC precursors, and the hPSC-KC precursors are induced to be mature into hPSC-KC cells; and / or the hPSC cells are differentiated into hPSC-FB cells, including mesoderm induced differentiation and fibroblast induced differentiation of the hPSC cells. The ready-to-use skin seed cell independent of a patient is prepared, the cell is directly added into a medium to be sprayed for repairing skin injury and reducing further injury of skin tissue of the patient, operation is convenient, rapid treatment is achieved, and the treatment effect is good. Meanwhile, the problems that autologous seed cells of patients are unstable in function and poor in regeneration capacity due to basic diseases are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a ready-to-use skin seed cell and a preparation method and application thereof. Background Art

[0002] Skin burns and trauma can easily lead to skin damage, especially in traffic accidents, industrial accidents, fires and other emergency situations. Promoting the regeneration of the patient's epidermal tissue as soon as possible is the key to repairing large-area skin burns or traumatic wounds. Conventional treatment often involves obtaining the patient's own healthy skin for treatment, but there may also be possible postoperative complications such as insufficient sources of healthy skin, poor survival of skin grafts, and damage to healthy tissue areas, which can delay treatment effects and even seriously affect treatment.

[0003] At present, many methods reconstruct the skin tissue of the affected area through in vitro expansion and transplantation of the patient's own cells, but these methods often take several weeks to obtain and expand the patient's own cells in vitro, and cannot play a timely and effective therapeutic role. Among them, Recell technology is a relatively effective technology that reconstructs skin tissue through autologous cell acquisition and transplantation, and has been used in the clinical treatment of large-area skin burns and trauma. This method obtains the patient's own epidermal tissue, digests it into a seed cell (mainly epidermal cells and fibroblasts) suspension, and then disperses it on the patient's wound surface by spraying. After a period of tissue growth, the sprayed seed cells proliferate in large quantities and eventually cover the entire wound surface to achieve burn treatment. Recell technology can accelerate epithelial regeneration and promote the repair and healing of skin wounds.

[0004] However, Recell technology still has the following problems, which may seriously limit its clinical application and therapeutic effect:

[0005] (1) The seed cells derived from patients may have poor functions, making it difficult to achieve skin tissue regeneration: For patients who are older or have underlying diseases (such as diabetes), the functions of their own epidermal cells may have been partially lost, and their survival and proliferation abilities after spraying are poor, making it difficult to quickly and comprehensively complete epidermal regeneration of the wound surface.

[0006] (2) Tissue digestion fluid may further damage the wound surface: In the Recell technology, digestive fluids such as pancreatic enzymes are needed to digest the patient's epidermal tissue into single cells for spraying. However, the sprayed pancreatic enzymes will severely digest and decompose the extracellular matrix in the dermis of the wound surface, which will hinder the efficiency of skin tissue regeneration to a certain extent.

[0007] (3) Complex operation: Since Recell technology relies on the processing of the patient's own tissue, there are many operation steps, including tissue extraction, digestion, spraying, etc., and the entire process is still relatively complicated.

[0008] (4) Healthy tissue from patients is required as a donor source. Summary of the invention

[0009] Based on this, the main purpose of the present invention is to provide a ready-to-use skin seed cell, which has the advantages of being cryopreservable, having a certain proliferation ability, having perfect cell functions and being ready to use.

[0010] Another object of the present invention is to provide a method for preparing the ready-to-use skin seed cells.

[0011] Another object of the present invention is to provide the use of the ready-to-use skin seed cells in preparing products for repairing skin damage.

[0012] To achieve the above object, the present invention adopts the following technical solution:

[0013] The present invention provides a ready-to-use skin seed cell, which uses human pluripotent stem cells (hPSC) as the source of the seed cell, and comprises keratinocytes (hPSC-KC) and / or fibroblasts (hPSC-FB) differentiated from the human pluripotent stem cells (hPSC).

[0014] Preferably, the type of human pluripotent stem cells is selected from one or more of the patient's own human pluripotent stem cells, wild-type human pluripotent stem cells, gene-edited embryonic stem cells, and human induced pluripotent stem cells.

[0015] The present invention also provides a method for preparing the ready-to-use skin seed cells, comprising the following steps:

[0016] (1) hPSC cell culture;

[0017] (2) the hPSC cells are differentiated into hPSC-KC cells, including the steps of differentiating the hPSC cells into hPSC-KC precursors and inducing the hPSC-KC precursors to mature into hPSC-KC cells; and / or

[0018] (3) The hPSC cells are differentiated into hPSC-FB cells, including the steps of mesoderm-inducing differentiation and fibroblast-inducing differentiation of the hPSC cells.

[0019] Preferably, in step (1), hPSC cells are seeded in a culture dish coated with extracellular matrix gel and cultured using human pluripotent stem cell culture medium. The culture medium is changed every day, and cell subculture begins when the cell confluence reaches 80%. On the day of subculture, a ROCK inhibitor (Y27632) is added to the culture medium at a final concentration of 5 μM, and culture is continued on the second day using a culture medium without Y27632.

[0020] Preferably, in step (2), the step of differentiating the hPSC cells into hPSC-KC precursors comprises:

[0021] (2-1) The hPSC cells were seeded in a culture dish coated with extracellular matrix gel at a ratio of 1:2 during passaging, and after passaging, the human pluripotent stem cell culture medium was supplemented with Y27632 at a final concentration of 10 μM;

[0022] (2-2) When the hPSC cells grow to 90-100% confluence, differentiation begins, and the culture medium is replaced with epidermal differentiation medium 1 containing 1 μM retinoic acid and 20 ng / mL BMP4, wherein the basal liquid components include: DMEM / F12 medium, knockout serum replacement, L-glutamine, non-essential amino acids, mercaptoethanol, antibiotics, or other commercial epidermal cell proliferation medium basal liquid, and the cells are cultured continuously for 5 days, with the culture medium replaced every day;

[0023] (2-3) On the 5th day of differentiation, the hPSC cells were re-seeded in a culture dish coated with extracellular matrix gel at a ratio of 1:2, and after subculturing, Y27632 at a final concentration of 10 μM was added to the epidermal differentiation medium 1;

[0024] (2-4) On the 6th day of differentiation, the medium was replaced with epidermal differentiation medium 2, which includes: DMEM / F12, N2 supplement, L-glutamine, non-essential amino acids, mercaptoethanol, and antibiotics. On the 6th and 7th days, 1 μM retinoic acid and 20 ng / mL BMP4 were continuously added to the medium. On the 8th day, retinoic acid and BMP4 were removed and replaced with 10 ng / mL EGF. The medium was changed every day until the 14th day of culture. The obtained hPSC-KC precursors were cryopreserved at a concentration of 1 M cell / mL using cryopreservation solution.

[0025] Preferably, in step (2), the step of inducing the hPSC-KC precursors to mature into hPSC-KC cells comprises:

[0026] (2-a) On day 14, the digested cells were subcultured into 6-well plates coated with extracellular matrix and cultured in epidermal differentiation medium 2 containing 10 ng / mL EGF. After subculturing, Y27632 was added to the culture medium at a final concentration of 10 μM;

[0027] (2-b) On day 15, Y27632 was removed and epidermal differentiation medium 2 containing 10 ng / mL EGF was added again. The culture medium was used for another 8 days, and the medium was changed every other day.

[0028] (2-c) On day 23, calcium chloride at a final concentration of 1.2 mM was added to epidermal differentiation medium 2, and the medium was changed every other day to begin inducing keratinocyte maturation. Mature hPSC-KC cells were obtained by culturing on day 30-45.

[0029] Preferably, in step (3), the step of inducing mesoderm differentiation of the hPSC cells comprises: subculturing the hPSC cells using a human pluripotent stem cell culture medium, and changing to a mesoderm differentiation culture medium on the next day (day 0), whose components include DMEM / F12, antibiotics, L-glutamine, ascorbic acid, and CHIR99021, and culturing for two days.

[0030] Preferably, in step (3), the step of inducing differentiation of hPSC-FB cells comprises: replacing the medium with fibroblast differentiation medium on the second day of differentiation, wherein the basal medium comprises DMEM medium, L-glutamine, antibiotics, and serum substitutes, and adding 100 ng / mL bFGF to the medium; further subculturing and amplifying the cells on the 20th day of differentiation, replacing the growth factors in the medium with 10 ng / mL bFGF and 10 ng / mL EGF to further stimulate cell proliferation, thereby obtaining hPSC-FB cells, and cryopreserving the hPSC-FB cells at a concentration of 1 M cells / mL using a freezing solution.

[0031] The present invention also provides use of the ready-to-use skin seed cells in preparing products for repairing skin damage.

[0032] Preferably, the product for repairing skin damage comprises the ready-to-use skin seed cells and a spray medium without digestive enzymes, and is used in an in vitro spraying manner.

[0033] The present invention also provides a product for repairing skin damage, comprising the ready-to-use skin seed cells and a spray medium without digestive enzymes.

[0034] Preferably, the digestive enzyme-free spraying medium is selected from serum-free DMEM medium or fibrin gel; when the serum-free DMEM medium is used as the spraying medium, the ready-to-use skin seed cells are directly resuspended in the medium to a final cell concentration of 500K / mL;

[0035] When the fibrin gel is used as a spraying medium, it includes a 20 mM HEPES solution containing a final concentration of 30 mg / mL fibrinogen, and a thrombin solution containing a final concentration of 1200 U / mL and a final concentration of 100 mM CaCl 2 of saline.

[0036] The present invention establishes a method for repairing skin damage based on ready-to-use skin seed cells and cell spraying, which has the following beneficial effects compared to the existing Recell technology:

[0037] 1. Prepare a ready-to-use skin seed cell that is independent of patient acquisition: hPSC cells are used as the source of seed cells, and they are differentiated into hPSC-KC cells and hPSC-FB cells that can be cryopreserved, have a certain proliferation capacity, and have complete cell functions, to prepare a large number of seed cells with complete functions that can be taken and used immediately, which does not rely on patients for seed cells. For example, "immune universal" human induced pluripotent stem cells with HLA gene knockout can be used as the source of skin cells. This ready-to-use, non-immunogenic allogeneic seed cell can replace the patient's own cells, without the need to obtain tissue from the patient, reducing further damage to the patient's skin tissue, and at the same time solving the problem of unstable function and poor regeneration ability of the patient's autologous seed cells caused by underlying diseases.

[0038] 2. Reduce damage to the patient's skin wound by removing digestive enzymes from the spraying medium: Establish a spraying medium without digestive enzymes for cell spraying, which does not rely on the patient's tissues and does not require digestive fluids such as pancreatic enzymes, and then construct a spraying medium without digestive enzymes that is conducive to the survival and retention of cells after spraying, thereby avoiding further damage to the patient's wound by the digestive fluid.

[0039] 3. Establish a simpler operation method than the Recell system: Use cryopreserved ready-to-use seed cells, and there is no need to obtain and digest the patient's tissue. There is no need to obtain and digest the patient's tissue. The cells can be directly added to the medium for spraying, which greatly simplifies the operation process, facilitates operation, and achieves rapid treatment.

[0040] 4. There is no need for donor skin tissue, reducing the possibility of damage to healthy tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The figure is a flow chart for preparing ready-to-use skin seed cells in the examples.

[0042] Figure 2 The figure shows the changes in cell morphology during the differentiation of hPSC-KC cells in the example.

[0043] Figure 3 : is the gene expression level of hPSC-KC cells in the example.

[0044] Figure 4 The figure shows the changes in cell morphology during the differentiation of hPSC-FB cells in the example.

[0045] Figure 5This is an immunofluorescence staining image of hPSC-FB cells in the example, red is the marker, and blue is the cell nucleus.

[0046] Figure 6 The figures show the survival of fibroblasts in the culture plate after being resuspended in a spraying medium based on DMEM culture medium and sprayed in the examples. Calcein AM: calcein AM; PI: propidium iodide.

[0047] Figure 7 This is the fibrin gel that is rapidly formed within one minute after spraying in the example.

[0048] Figure 8 The figure shows the survival of fibroblasts in fibrinogen gel after spraying in the example. CalceinAM: calcein AM; PI: propidium iodide. DETAILED DESCRIPTION

[0049] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. It should be pointed out that for ordinary technicians in this field, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention.

[0050] Example 1

[0051] According to Figure 1 The process shown is to prepare ready-to-use skin seed cells, the steps are as follows:

[0052] (1) hPSC cell culture:

[0053] hPSC cells (including autologous, wild-type, gene-edited embryonic stem cells, human induced pluripotent stem cells and other types of human pluripotent stem cells) were inoculated in a culture dish with extracellular matrix gel, and cultured in human pluripotent stem cell medium, which was changed every day. When the cell confluence reached 80%, the cells were subcultured. On the day of subculture, the ROCK inhibitor Y27632 was added to the culture medium at a final concentration of 5 μM, and the culture was continued on the second day using a medium without Y27632.

[0054] (2) Human pluripotent stem cell-derived keratinocytes (hPSC-KC)

[0055] (2-1) Differentiation of hPSCs into hPSC-KC precursors:

[0056] During passaging, hPSCs were seeded in a culture dish coated with Matrigel at a ratio of 1:2, and after passaging, the human pluripotent stem cell culture medium was supplemented with Y27632 at a final concentration of 10 μM.

[0057] When hPSC cells grow to 90-100% confluence, differentiation begins: the existing culture medium is replaced with epidermal differentiation medium 1 containing 1 μM retinoic acid and 20 ng / mL BMP4, and its basal liquid components include: DMEM / F12 medium, knockout serum replacement, L-glutamine, non-essential amino acids, mercaptoethanol, antibiotics (or other commercial epidermal cell proliferation medium basal liquid), and the epidermal differentiation medium is used for continuous culture for 5 days, and the culture medium is replaced every day.

[0058] On the 5th day of differentiation, the cells were re-seeded in a culture dish coated with extracellular matrix at a ratio of 1:2, and Y27632 was added to the epidermal differentiation medium 1 at a final concentration of 10 μM after passaging.

[0059] On the 6th day of differentiation, the medium was changed to epidermal differentiation medium 2, which includes: DMEM / F12, N2 supplement, L-glutamine, non-essential amino acids, mercaptoethanol, antibiotics, 1 μM retinoic acid and 20 ng / mL BMP4 were continuously added to the medium on the 6th and 7th days, retinoic acid and BMP4 were removed on the 8th day, and replaced with 10 ng / mL EGF, and the medium was changed every day until the 14th day of culture. The hPSC-KC precursors were cryopreserved at a concentration of 1 M cells / mL using cryopreservation solution.

[0060] (2-2) hPSC-KC induction maturation:

[0061] On day 14, the digested cells were passaged into 6-well plates coated with extracellular matrix and cultured in epidermal differentiation medium 2 containing 10 ng / mL EGF. After passage, Y27632 was added to the culture medium at a final concentration of 10 μM.

[0062] On day 15, Y27632 was removed and epidermal differentiation medium 2 containing 10 ng / mL EGF was added again. The culture medium was used to continue culturing for 8 days, and the medium was changed every other day (or other commercial epidermal cell maturation medium was selected).

[0063] On day 23, calcium chloride was supplemented to a final concentration of 1.2 mM into epidermal differentiation medium 2, and the medium was changed every other day to begin inducing keratinocyte maturation. Mature hPSC-KCs were obtained on day 30-45 of culture.

[0064] (3) Human pluripotent stem cell-derived fibroblasts (hPSC-FB)

[0065] (3-1) Mesoderm differentiation of hPSCs:

[0066] hPSC cells were subcultured with human pluripotent stem cell culture medium, and the next day (day 0) the medium was replaced with mesoderm differentiation medium, whose components included DMEM / F12, antibiotics, L-glutamine, ascorbic acid, and CHIR99021, and cultured for two days (alternatives include other commercial mesoderm induction medium).

[0067] (3-2) Fibroblast differentiation induction:

[0068] On the second day of differentiation, the medium was replaced with fibroblast differentiation medium, whose basal solution included DMEM medium, L-glutamine, antibiotics, and serum replacement. At the same time, 100 ng / mL bFGF was added to the medium, and further passage and expansion were performed on the 20th day of differentiation (or other commercial fibroblast induction medium was selected). During the expansion process, the growth factors in the medium were replaced with 10 ng / mL bFGF and 10 ng / mL EGF to further stimulate cell proliferation, obtain hPSC-FB (or select other commercial fibroblast proliferation medium), and cryopreserved hPSC-FB at a concentration of 1 M cells / mL using freezing solution.

[0069] (4) Spraying seed cells

[0070] (4-1) Obtaining frozen hPSC-KC precursors and hPSC-FB cells: Thaw and revive the cells in a 37°C water bath, remove the supernatant after centrifugation, and replace the cryopreservation medium with DMEM medium containing 10% serum.

[0071] (4-2) While reviving the cells, prepare the spraying medium: the spraying medium uses serum-free DMEM culture medium (or other commercial culture medium base fluids, such as DMEM / F12, M199, etc.), or uses a fibrin gel-based spraying medium (or a hydrogel biomaterial including type I collagen, hyaluronic acid, methacryloyl gelatin, etc.). The fibrin spraying medium includes a 20 mM HEPES solution containing a final concentration of 30 mg / mL fibrinogen (spraying medium 1), and a thrombin solution containing a final concentration of 1200 U / mL and a final concentration of 100 mM CaCl 2 of normal saline (spraying medium 2).

[0072] Mix hPSC-KC precursors and hPSC-FB cells in a certain ratio as needed and resuspend the cells in spraying medium:

[0073] If serum-free DMEM is used as the spraying medium, resuspend the seeded cells directly in the medium to a final cell concentration of 500K / mL.

[0074] If fibrin gel is used as the spraying medium, resuspend the seeded cells in spraying medium 1 to a final cell concentration of 500K / mL.

[0075] (4-3) Spraying cells:

[0076] The spraying tool is a press spray bottle (or an atomizing nasal sprayer, an air pump-type small spray gun, or other tools that can spray atomized liquids), and the spraying pressure is not higher than 15psi.

[0077] If serum-free DMEM medium is used as the spraying medium, add the DMEM cell suspension into the spraying device and spray directly. Every 500K cells can be sprayed on about 10cm 2 on the area.

[0078] If fibrin gel is used as the spraying medium, prepare two spraying tools and add the same volume of spraying medium 1 (containing resuspended seed cells) and spraying medium 2 respectively. When spraying, first spray the gel reaction solution 2 onto the matrix surface (such as the patient's skin wound or the surface of the culture dish used for the experiment), then spray the same volume of spraying medium 1 containing resuspended cells, and then spray back and forth.

[0079] Example 2

[0080] 2.1 Effect of hPSC-derived keratinocyte precursors (hPSC-KC)

[0081] (1) hPSC-KC is differentiated from wild-type human induced pluripotent stem cells. Figure 2 The morphology of the cells at different time points of differentiation is displayed, where the 14th day (D14) is the hPSC-KC precursor cells used for cell spraying; the 15th and 20th days (D15 and D20) are the process of hPSC-KC cell maturation culture.

[0082] (2) The obtained hPSC-KC cells expressed a series of keratinocyte markers (KRT18, KRT14, KRT1, P63, E-cadherin, IVL) through qRT-PCR, and their expression levels were much higher than those of undifferentiated human pluripotent stem cells (hiPSC). Among them, the mature keratinocyte marker IVL was the highest in hPSC-KC differentiated to day 23. This indicates that hPSC-K has the phenotype of keratinocytes and can be further induced to mature ( Figure 3 ).

[0083] 2.2 Effect of hPSC-derived fibroblasts (hPSC-FB)

[0084] hPSC-FB is differentiated from wild-type human induced pluripotent stem cells. Figure 4The morphology of cells at different time points of differentiation is shown. On the sixth day of differentiation (D6), the morphology of cells changes significantly. Through continuous differentiation and expansion, fibroblasts with typical spindle morphology appear in the first and third generations (P1, P3) after expansion.

[0085] The above hPSC-FB expressed typical fibroblast markers, including α-SMA, Collagen I, Collagen III, Laminin beta 1, Fibronectin ( Figure 5 ).

[0086] 2.3 Effect of spraying cells

[0087] Using a spray medium based on DMEM culture medium and a spray bottle, fibroblasts were sprayed on the culture dish to test the effect of spraying on cell activity. Through the cell live-death staining experiment (calcein-propidium iodide staining), the results showed that almost all cells can survive well ( Figure 6 ).

[0088] At the same time, using a fibrinogen-based spray medium and a spray bottle, fibroblasts were sprayed onto the culture dish, and the cells were encapsulated and survived in the fibrinogen gel. Cell live-dead staining showed that most of the cells were alive ( Figure 7 and 8 ).

[0089] In addition, the present invention also relates to a method for spraying / applying / applying / wound delivery of skin seed cells, using frozen hPSC-KC precursors and hPSC-FB as seed cell sources for repairing damaged skin. First, the cells are thawed and revived, and the supernatant is removed by centrifugation, and the cryopreservation solution is replaced with a culture medium. At the same time, a medium is prepared, and a commercial culture medium or a cell spraying medium is selected. A hydrogel material medium such as a fibrin gel can also be used. After the hPSC-KC precursor and hPSC-FB are mixed as needed, the cells are resuspended to the corresponding concentration according to the type of medium. When applying the cells, a spraying tool can be used for spraying, or the cells can be evenly applied to the wound surface by smearing, microneedle injection, etc., so as to achieve the repair of skin damage. The operation is flexible and diverse, and the problem of unstable function of the patient's autologous seed cells can be solved.

[0090] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A ready-to-use skin seed cell, characterized in that: Human pluripotent stem cells (hPSC) are used as the source of seed cells, including keratinocytes (hPSC-KC) and / or fibroblasts (hPSC-FB) differentiated from the hPSC cells.

2. The ready-to-use skin seed cells according to claim 1, characterized in that: The type of human pluripotent stem cells is selected from one or more of the patient's own human pluripotent stem cells, wild-type human pluripotent stem cells, gene-edited embryonic stem cells, and human induced human pluripotent stem cells.

3. The method for preparing the ready-to-use skin seed cells according to claim 1 or 2, characterized in that: The following steps are involved: (1) hPSC cell culture; (2) the hPSC cells are differentiated into hPSC-KC cells, including the steps of differentiating the hPSC cells into hPSC-KC precursors and inducing the hPSC-KC precursors to mature into hPSC-KC cells; and / or (3) The hPSC cells are differentiated into hPSC-FB cells, including the steps of mesoderm-inducing differentiation and fibroblast-inducing differentiation of the hPSC cells.

4. The method for preparing ready-to-use skin seed cells according to claim 3, characterized in that: In step (1), hPSC cells are seeded in a culture dish coated with extracellular matrix gel and cultured using human pluripotent stem cell culture medium. The culture medium is changed every day. When the cell confluence reaches 80%, subculturing begins. On the day of subculturing, Y27632 is added to the culture medium at a final concentration of 5 μM. On the second day, culture is continued using a culture medium without Y27632.

5. The method for preparing ready-to-use skin seed cells according to claim 3, characterized in that: In step (2), the step of differentiating the hPSC cells into hPSC-KC precursors comprises: (2-1) The hPSC cells were seeded in a culture dish coated with extracellular matrix gel at a ratio of 1:2 during passaging, and after passaging, the human pluripotent stem cell culture medium was supplemented with Y27632 at a final concentration of 10 μM; (2-2) When the hPSC cells grow to 90-100% confluence, differentiation begins, and the culture medium is replaced with epidermal differentiation medium 1 containing 1 μM retinoic acid and 20 ng / mL BMP4, wherein the basal liquid components include: DMEM / F12 medium, knockout serum replacement, L-glutamine, non-essential amino acids, mercaptoethanol, antibiotics, or other commercial epidermal cell proliferation medium basal liquid, and the cells are cultured continuously for 5 days, with the culture medium replaced every day; (2-3) On the 5th day of differentiation, the hPSC cells were re-seeded in a culture dish coated with extracellular matrix gel at a ratio of 1:2, and after subculturing, Y27632 at a final concentration of 10 μM was added to the epidermal differentiation medium 1; (2-4) On the 6th day of differentiation, the medium was replaced with epidermal differentiation medium 2, which includes: DMEM / F12, N2 supplement, L-glutamine, non-essential amino acids, mercaptoethanol, and antibiotics. On the 6th and 7th days, 1 μM retinoic acid and 20 ng / mL BMP4 were continuously added to the medium. On the 8th day, retinoic acid and BMP4 were removed and replaced with 10 ng / mL EGF. The medium was changed every day until the 14th day of culture. The obtained hPSC-KC precursors were cryopreserved at a concentration of 1 M cells / mL using cryopreservation solution; The step of inducing hPSC-KC precursors to mature into hPSC-KC cells comprises: (2-a) On day 14, the digested cells were subcultured into 6-well plates coated with extracellular matrix and cultured in epidermal differentiation medium 2 containing 10 ng / mL EGF. After subculturing, Y27632 was added to the culture medium at a final concentration of 10 μM; (2-b) On day 15, Y27632 was removed and epidermal differentiation medium 2 containing 10 ng / mL EGF was added again. The culture medium was used for another 8 days, and the medium was changed every other day. (2-c) On day 23, calcium chloride at a final concentration of 1.2 mM was added to epidermal differentiation medium 2, and the medium was changed every other day to begin inducing keratinocyte maturation. Mature hPSC-KC cells were obtained by culturing on day 30-45.

6. The method for preparing ready-to-use skin seed cells according to claim 3, characterized in that: In step (3), the step of inducing mesoderm differentiation of the hPSC cells comprises: subculturing the hPSC cells using a human pluripotent stem cell culture medium, and replacing it with a mesoderm differentiation culture medium the next day, whose components include DMEM / F12, antibiotics, L-glutamine, ascorbic acid, and CHIR99021, and culturing for two days.

7. The method for preparing ready-to-use skin seed cells according to claim 3, characterized in that: In step (3), the step of inducing differentiation of hPSC-FB cells includes: replacing the medium with fibroblast differentiation medium on the second day of differentiation, wherein the basal medium includes DMEM medium, L-glutamine, antibiotics, and serum substitutes, and adding 100 ng / mL bFGF to the medium; further subculturing and amplification on the 20th day of differentiation, the growth factors in the medium are replaced with 10 ng / mL bFGF and 10 ng / mL EGF to further stimulate cell proliferation, thereby obtaining hPSC-FB cells, and freezing and preserving the hPSC-FB cells at a concentration of 1 M cells / mL using a freezing solution.

8. Use of the ready-to-use skin seed cells according to claim 1 or 2 in preparing products for repairing skin damage.

9. The use according to claim 8, characterized in that: The product for repairing skin damage comprises the ready-to-use skin seed cells and a spray medium without digestive enzymes, and is used in an in vitro spraying manner.

10. A product for repairing skin damage, characterized in that: Comprising the ready-to-use skin seed cells according to claim 1 or 2 and a spray medium without digestive enzymes; The digestive enzyme-free spraying medium is selected from serum-free DMEM culture medium or fibrin gel; When the serum-free DMEM medium is used as a spraying medium, the ready-to-use skin seed cells are directly resuspended in the medium to a final cell concentration of 500K / mL; When the fibrin gel is used as a spraying medium, it includes a 20 mM HEPES solution containing a final concentration of 30 mg / mL fibrinogen, and a physiological saline solution containing a final concentration of 1200 U / mL thrombin and a final concentration of 100 mM CaCl2.

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