A method for preparing keratinocytes derived from pluripotent stem cells and its application

Abstract

This invention provides a method for preparing keratinocytes derived from pluripotent stem cells and its application. The preparation method includes: passage of pluripotent stem cells; pretreatment of the passaged pluripotent stem cells with multiple inhibitors; culturing the pretreated pluripotent stem cells in an induction medium to obtain first pluripotent stem cells, wherein the induction medium contains BMP4 and retinoic acid compounds; passage of the first pluripotent stem cells into a first keratinocyte culture medium to obtain second pluripotent stem cells; and culturing the second pluripotent stem cells in a second keratinocyte culture medium to obtain keratinocytes. This invention can achieve high differentiation efficiency, short preparation cycle, strong stability, and good reproducibility in the preparation of keratinocytes, and can construct functional three-dimensional full-thickness tissue-engineered skin, thereby breaking through the existing technical bottlenecks and meeting the application needs of clinical skin wound repair.

Description

Technical Field

[0001] This invention relates to the field of cell engineering technology, and more specifically, to a method for preparing keratinocytes derived from pluripotent stem cells and their application. Background Technology

[0002] The skin is the largest organ in the human body and the first physiological barrier against external physical, chemical, and biological stimuli. When burns or other injuries cause full-thickness skin loss, if the wound cannot be repaired in time, it is very easy to cause complications such as wound infection and massive loss of body fluids, which can endanger the patient's life in severe cases.

[0003] Currently, autologous skin grafting is the mainstream clinical treatment for large-area skin trauma and burns, and it is also the gold standard in the field of wound repair. However, this method has significant limitations: on the one hand, the number of healthy skin cells from patients with large-area trauma is severely insufficient to meet the wound coverage requirements; on the other hand, the skin harvesting process can cause secondary trauma to patients. While xenogeneic or allogeneic skin can temporarily cover the wound and provide short-term protection, it carries a strong risk of immune rejection and cross-transmission of diseases, greatly limiting its clinical application. Therefore, constructing tissue-engineered skin substitutes with complete physiological functions and capable of large-scale preparation has become an urgent technical challenge to be solved in the field of skin wound repair.

[0004] Keratinocytes are the most important functional cells in the epidermis, playing a central role in wound re-epithelialization and skin tissue remodeling, and are key seed cells for constructing tissue-engineered skin. Traditional techniques primarily use primary keratinocytes, but their availability is limited, their in vitro proliferation capacity is weak, making large-scale expansion difficult and unsuitable for large-scale clinical applications. Pluripotent stem cells (PSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), possess unlimited self-renewal capacity and multi-lineage directed differentiation potential, making them ideal seed cells for preparing standardized, regenerable keratinocytes.

[0005] Among related technologies, the differentiation of pluripotent stem cells into keratinocytes suffers from issues with poor purity and function of epidermal cells. Furthermore, existing differentiation processes have long overall cycles, with conventional culture and differentiation taking over 30 days and exhibiting low differentiation efficiency. To purify the target cells, additional auxiliary processes such as low-calcium screening and flow cytometry sorting are required to obtain keratinocyte-like cells expressing KRT5 and KRT14, further increasing operational complexity and preparation costs, severely hindering the clinical translation of this technology. Summary of the Invention

[0006] This invention aims to provide a method for preparing keratinocytes derived from pluripotent stem cells and its application. This method achieves high differentiation efficiency, short preparation cycle, strong stability and good reproducibility of keratinocytes by effectively inhibiting trophoblast lineage differentiation and specifically inducing epidermal ectoderm development. Furthermore, it can construct functional three-dimensional full-thickness tissue-engineered skin based on the obtained cells, thereby breaking through the existing technical bottlenecks and meeting the application needs of clinical skin wound repair.

[0007] To address the above problems, a first aspect of the present invention provides a method for preparing keratinocytes derived from pluripotent stem cells, comprising the following steps:

[0008] Pluripotent stem cells are passaged, and the passaged pluripotent stem cells are pretreated with multiple inhibitors. After the pretreatment, pretreated pluripotent stem cells are obtained. The multiple inhibitors include SMAD2 / 3 pathway inhibitors, SMAD1 / 5 / 9 pathway inhibitors and FGF2 pathway inhibitors.

[0009] The pretreated pluripotent stem cells were cultured in an induction medium to obtain first pluripotent stem cells, wherein the induction medium contained BMP4 and retinoic acid compounds.

[0010] The first pluripotent stem cells were passaged into a first keratinocyte culture medium at a preset ratio and cultured. After the first pluripotent stem cells reached a high density, the first pluripotent stem cells were collected and passaged into a first keratinocyte culture medium at a preset ratio to obtain second pluripotent stem cells.

[0011] The second pluripotent stem cells were cultured in a second keratinocyte culture medium to obtain keratinocytes;

[0012] Both the first keratinocyte culture medium and the second keratinocyte culture medium contain calcium ions, with the calcium ion concentration in the first keratinocyte culture medium being higher than that in the second keratinocyte culture medium.

[0013] Furthermore, the multiple inhibitors pretreat the passaged pluripotent stem cells for 12 to 48 hours;

[0014] The SMAD2 / 3 pathway inhibitor is selected from at least one of SB431542, A8301 and pharmaceutically acceptable salts thereof, and the concentration of the SMAD2 / 3 pathway inhibitor used is from 1 μmol / L to 20 μmol / L.

[0015] The SMAD1 / 5 / 9 pathway inhibitor is selected from at least one of LDN193189 and its pharmaceutically acceptable salts, and the concentration of the SMAD1 / 5 / 9 pathway inhibitor used is from 10 nmol / L to 1000 nmol / L;

[0016] The FGF2 pathway inhibitor is selected from at least one of PD0325901, PD173074, SU5402 and their pharmaceutically acceptable salts, and the concentration of the FGF2 pathway inhibitor used is from 10 nmol / L to 1000 nmol / L.

[0017] Furthermore, the concentration of BMP4 in the induction medium is from 1 ng / mL to 100 ng / mL, and the concentration of the retinoic acid compound is from 0.1 μmol / L to 10 μmol / L;

[0018] The retinoic acid compound is selected from at least one of retinoic acid, TTNPB, and pharmaceutically acceptable salts thereof.

[0019] Furthermore, the pretreated pluripotent stem cells were cultured in induction medium for 5 to 9 days, with the induction medium being replaced with fresh medium every 2 days.

[0020] Furthermore, the first pluripotent stem cells are passaged into the first keratinocyte culture medium at a preset ratio and cultured for 5 to 10 days. The first pluripotent stem cells are then collected and passaged into the first keratinocyte culture medium at a preset ratio and cultured for 1 day.

[0021] The preset ratio is 1:2 to 1:6, and the concentration of calcium ions in the first keratinocyte culture medium is 1 mmol / L.

[0022] Furthermore, the second pluripotent stem cells were cultured in a second keratinocyte culture medium for 5 to 10 days;

[0023] The concentration of calcium ions in the second keratinocyte culture medium was 0.01 mmol / L to 0.1 mmol / L.

[0024] A second aspect of the present invention provides a 3D full-thickness skin model comprising keratinocytes prepared by the preparation method described in the first aspect.

[0025] Furthermore, the 3D full-thickness skin model is formed by seeding the keratinocytes onto the surface of a dermal substitute and then culturing them at an air-liquid interface. The seeding density of the keratinocytes is 2 × 10⁻⁶ cells per square centimeter. 4 Up to 1×10 6 Each cell.

[0026] Furthermore, the 3D full-thickness skin model shown also includes a dermal scaffold and fibroblasts;

[0027] The dermal scaffold is selected from any one or more combinations of collagen scaffolds, decellularized dermal scaffolds, and synthetic polymer scaffolds; the fibroblasts are selected from primary dermal fibroblasts, mesenchymal stem cells, or fibroblasts differentiated from mesenchymal stem cells.

[0028] The third aspect of the invention provides the use of keratinocytes prepared by the method described in the first aspect, or the use of a 3D full-thickness skin model as described in the second aspect, in at least one of the following:

[0029] To prepare drugs or medical devices for treating skin injuries;

[0030] To prepare an in vitro model for evaluating the efficacy of cosmetics;

[0031] Prepare in vitro models for drug screening.

[0032] The method for preparing keratinocytes derived from pluripotent stem cells described in this invention pretreats pluripotent stem cells with multiple inhibitors before inducing differentiation with BMP4 and retinoic acid compounds. This pretreatment inhibits the expression of the trophoblast characteristic gene CGB, thus blocking trophoblast lineage differentiation at its source before BMP4 and retinoic acid treatment. After the pluripotent stem cells are directionally induced to differentiate by BMP4 and retinoic acid compounds, a stepwise calcium ion concentration regulation strategy combined with continuous passage culture is used. First, culturing in a keratinocyte culture medium with a high calcium ion content promotes cell proliferation, while culturing in a keratinocyte culture medium with a low calcium ion content maintains cell stemness and promotes maturation. This enables rapid and large-scale preparation of keratinocytes. Compared with existing technologies, this invention can shorten the differentiation cycle by approximately 30% and significantly increase cell yield. Furthermore, the preparation method provided by this invention is applicable to different types of pluripotent stem cell lines and exhibits good stability and reproducibility.

[0033] The 3D full-thickness skin model of this invention involves seeding keratinocytes onto the surface of a dermal substitute and culturing them at the gas-liquid interface. This simulates the in vivo gas-liquid interface environment of skin tissue, promoting keratinocyte differentiation and improving skin barrier function, thereby constructing a structurally stable 3D full-thickness skin model. The 3D full-thickness skin constructed in this invention can not only be used in clinical applications for wound repair such as burns, but also serve as a standardized in vitro model in cosmetics and drug development. Compared with traditional animal experiments and primary cell models, the 3D full-thickness skin model provided in this invention has the following advantages:

[0034] (1) Reproducibility and standardization: The 3D full-thickness skin model provided in this invention uses pluripotent stem cells as the starting cells, which can establish a standardized cell bank with small batch-to-batch differences. This solves the problem of large differences and limited sources of primary cell donors, and provides a stable and reliable testing platform for cosmetic efficacy evaluation and drug screening.

[0035] (2) Significantly promotes wound repair: The 3D full-thickness skin model provided in this invention is constructed using keratinocytes, which have good wound repair capabilities. When this 3D skin model is used for wound repair such as burns, the regenerative capacity of keratinocytes can be used to effectively promote wound re-epithelialization and significantly improve the quality of skin healing.

[0036] (3) In line with the EU’s ban on animal testing: The 3D full-thickness skin model provided in this invention fully complies with the relevant provisions of the EU cosmetics regulations prohibiting animal testing. The 3D full-thickness skin model is constructed using keratinocytes and can reproduce the physiological structure of natural skin. It not only meets the regulatory requirements for safety testing of skin irritation, corrosiveness, phototoxicity, etc., but also has extremely high prospects for industrialization and promotion.

[0037] (4) Complete skin barrier function: The 3D full-layer skin model provided in this invention has a complete layered epidermal structure and stratum corneum, which can simulate the complete skin epidermal structure and its barrier function;

[0038] (5) Scalability and customization: Due to the gene-editable characteristics of pluripotent stem cells, this embodiment can also construct specific hereditary skin disease models (such as ichthyosis and epidermolysis bullosa) through gene modification for screening and mechanism research of rare skin disease drugs, providing a new in vitro research platform for precision medicine. Attached Figure Description

[0039] Figure 1 This is a process flow diagram for preparing keratinocytes provided in an embodiment of the present invention;

[0040] Figure 2 The diagram shows the verification results of the preparation method provided in the embodiments of the present invention applied in different cell lines.

[0041] Figure 3 This is a functional verification diagram of the iKERA keratinocytes obtained in the embodiments of the present invention;

[0042] Figure 4 This is an in vivo functional verification diagram of iKERA keratinocytes prepared in this embodiment of the invention for wound repair.

[0043] Figure 5 This is a phylogenetic analysis diagram of traditional preparation methods;

[0044] Figure 6 This is a schematic diagram comparing the phylogenetic differentiation pathways of the traditional preparation method and the preparation method of this embodiment;

[0045] Figure 7 This is a schematic diagram illustrating the preparation of keratinocytes (iKERA) during cell differentiation in an embodiment of the present invention.

[0046] Figure 8 These are diagrams illustrating different methods used in embodiments of the present invention to promote keratinocyte differentiation.

[0047] Figure 9 This is a schematic diagram illustrating the staged calcium regulation that promotes keratinocyte differentiation in an embodiment of the present invention. Detailed Implementation

[0048] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, specific embodiments of this application will be described in detail below with reference to the accompanying drawings.

[0049] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0050] Furthermore, the terms "comprising," "including," "containing," and "having" are non-restrictive and can refer to the addition of other steps and components that do not affect the results. Unless otherwise specified, all materials, equipment, and reagents are commercially available.

[0051] Furthermore, although this application describes each step in the preparation process in the form of S110, S120, S130 and S140, this description is only for ease of understanding. The forms such as S110, S120, S130 and S140 do not indicate a limitation on the order of the steps.

[0052] Combination Figure 1 As shown, a first aspect of this application provides a method for preparing keratinocytes derived from pluripotent stem cells, comprising the following steps:

[0053] Step S110: Passage the pluripotent stem cells and pretreat them with multiple inhibitors. After pretreatment, pretreated pluripotent stem cells are obtained. The multiple inhibitors include SMAD2 / 3 pathway inhibitors, SMAD1 / 5 / 9 pathway inhibitors and FGF2 pathway inhibitors.

[0054] Specifically, pluripotent stem cells are passaged, and the passaged pluripotent stem cells are added to E8 medium for pretreatment for 12 to 48 hours. After pretreatment, pretreated pluripotent stem cells are obtained. The E8 medium contains multiple inhibitors.

[0055] In related technologies, classic pluripotent stem cell differentiation into keratinocytes typically employs BMP4 and retinoic acid (RA) as core inducing factors. However, the inventors of this application have discovered for the first time that BMP4 simultaneously drives cell differentiation into the trophoblast lineage during the induction of differentiation, causing cells to express trophoblast-specific markers such as CGB and GCM1, resulting in an incorrect differentiation pathway and severely affecting the purity and biological function of epidermal cells. To address this issue, this application pretreats passaged pluripotent stem cells with a combination of multiple inhibitors before BMP4 and RA induction. Among these, the SMAD2 / 3 pathway inhibitor inhibits extraembryonic trophoblast and reduces the expression of the related gene CGB, while promoting epidermal ectoderm and increasing the expression of the related gene TP63. The SMAD1 / 5 / 9 pathway inhibitor, in combination with the SMAD2 / 3 pathway inhibitor, can further inhibit extraembryonic trophoblast and reduce the expression of the related gene CGB. Based on the inhibitors of the SMAD1 / 5 / 9 and SMAD2 / 3 pathways, FGF2 pathway inhibitors further promote epidermal ectoderm lineage differentiation and increase or maintain the expression of the related gene TP63. In this application, by using a multi-inhibitor combination including SMAD2 / 3, SMAD1 / 5 / 9, and FGF2 pathway inhibitors, the expression of the trophoblast characteristic gene CGB can be significantly suppressed, thereby blocking trophoblast lineage differentiation at its source.

[0056] In this embodiment, the SMAD2 / 3 pathway inhibitor is selected from at least one of SB431542, A8301, and pharmaceutically acceptable salts thereof; the SMAD1 / 5 / 9 pathway inhibitor is selected from at least one of LDN193189 and pharmaceutically acceptable salts thereof; and the FGF2 pathway inhibitor is selected from at least one of PD0325901, PD173074, SU5402, and pharmaceutically acceptable salts thereof. Preferably, the SMAD2 / 3 pathway inhibitor is selected from SB431542, the SMAD1 / 5 / 9 pathway inhibitor is selected from LDN193189, and the FGF2 pathway inhibitor is selected from PD0325901.

[0057] Based on the above embodiments, as an optional implementation, the concentration of the SMAD2 / 3 pathway inhibitor is 1 μmol / L to 20 μmol / L; the concentration of the SMAD1 / 5 / 9 pathway inhibitor is 10 nmol / L to 1000 nmol / L; and the concentration of the FGF2 pathway inhibitor is 10 nmol / L to 1000 nmol / L. Therefore, limiting the concentration of the SMAD2 / 3 pathway inhibitor within the above ranges avoids the situation where the concentration of the SMAD2 / 3 pathway inhibitor is too low, which would be insufficient to inhibit the extraembryonic trophoblast and reduce the expression of the related gene CGB, and promote the epidermal ectoderm and increase the expression of the related gene TP63. It also avoids the situation where the concentration of the SMAD2 / 3 pathway inhibitor is too high, which would increase differentiation costs and cause cytotoxicity. Limiting the concentration of SMAD1 / 5 / 9 pathway inhibitors within the aforementioned range avoids situations where the concentration is too low, failing to inhibit extraembryonic trophoblast differentiation and reduce the expression of the related gene CGB, and avoids situations where the concentration of SMAD1 / 5 / 9 pathway inhibitors is too high, inhibiting epidermal ectoderm differentiation and suppressing the expression of the related gene TP63. Similarly, limiting the concentration of FGF2 pathway inhibitors within the aforementioned range avoids situations where the concentration is too low, resulting in a decreased effect on promoting epidermal ectoderm lineage differentiation and increasing or maintaining the expression of the related gene TP63, and avoids situations where the concentration is too high, leading to slower cell proliferation and decreased yield.

[0058] Based on the above embodiments, as a preferred embodiment, the concentration of the SMAD2 / 3 pathway inhibitor is 10 μmol / L; the concentration of the SMAD1 / 5 / 9 pathway inhibitor is 100 nmol / L; and the concentration of the FGF2 pathway inhibitor is 100 nmol / L. In this embodiment, the E8 (TeSR-E8™ (Essential 8™ Medium)) medium contains not only the above-mentioned multiple inhibitors but also basic components for maintaining pluripotent stem cells in an undifferentiated state. Those skilled in the art can determine the basic components according to the actual situation, and this embodiment does not further limit them.

[0059] Based on the above embodiments, as a preferred embodiment, the passaged pluripotent stem cells are pretreated with multiple inhibitors for 24 hours. Therefore, the pretreatment time is within the above range, ensuring that the multiple inhibitors adequately pretreat the pluripotent stem cells.

[0060] In this embodiment, pluripotent stem cells include human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). hESCs include the hESC H1 cell line and the hESC H9 cell line, and iPSCs include the hiPSC ND1 cell line.

[0061] In this embodiment, the following method can be used to passage pluripotent stem cells: pluripotent stem cells cultured in Matrigel-coated culture plates are passaged with EDTA solution and then seeded into new Matrigel-coated culture plates at an appropriate ratio (which can be determined by those skilled in the art based on actual conditions). Maintenance pluripotent stem cell culture medium is added for continued culture. After the cells re-attach and spread, subsequent experiments or routine medium changes are performed for maintenance.

[0062] Step S120: The pretreated pluripotent stem cells are cultured in an induction medium to obtain the first pluripotent stem cells. The induction medium contains BMP4 and retinoic acid compounds.

[0063] Specifically, the pretreated pluripotent stem cells were transferred to an induction medium and cultured for 5 to 9 days, with the medium being replaced with fresh medium every 2 days to obtain the first pluripotent stem cells. In this embodiment, by pretreating the pluripotent stem cells and then inducing them with BMP4 and retinoic acid compounds, it is beneficial to induce the pluripotent stem cells to differentiate into keratinocytes and to block trophoblastic lineage differentiation at the source.

[0064] Based on the above embodiments, as an optional implementation, the concentration of BMP4 in the induction culture medium is from 1 ng / mL to 100 ng / mL, and the concentration of the retinoic acid compound is from 0.1 μmol / L to 10 μmol / L; wherein the retinoic acid compound is selected from at least one of retinoic acid (RA), TTNPB, and pharmaceutically acceptable salts thereof. Therefore, concentrations of BMP4 and retinoic acid compounds within the above ranges are beneficial for promoting the induced differentiation of pluripotent stem cells into keratinocytes.

[0065] Based on the above embodiments, as a preferred embodiment, the concentration of BMP4 in the induction medium is 20 ng / mL, and the retinoic acid compound is RA at a concentration of 1 μmol / L, or the retinoic acid compound is TTNPB at a concentration of 0.1 μmol / L to 1 μmol / L. Therefore, the concentrations of each component in the induction medium are within the above ranges, which is beneficial for improving differentiation efficiency and stability. In this embodiment, in addition to BMP4 and the retinoic acid compound, the induction medium also contains basal medium E6 (Essential 6™ Flex Medium (Cat. A1516401)). Those skilled in the art can determine the composition of E6 by referring to existing technologies; this embodiment does not further limit this.

[0066] Based on the above embodiments, as a preferred embodiment, the pretreated pluripotent stem cells are cultured in an induction medium for 7 days. Therefore, culturing the pretreated pluripotent stem cells in the induction medium for 7 days optimizes the efficiency of BMP4 and retinoic acid-induced epidermal ectoderm differentiation and results in more stable differentiation.

[0067] Step S130: Passage the first pluripotent stem cells into the first keratinocyte culture medium at a preset ratio and culture them. After the first pluripotent stem cells reach a high density, collect the first pluripotent stem cells and continue to passage them into the first keratinocyte culture medium at a preset ratio to obtain the second pluripotent stem cells.

[0068] Specifically, first pluripotent stem cells are passaged in a first keratinocyte culture medium at a predetermined ratio and cultured for 5 to 10 days. Once the first pluripotent stem cells reach a high density (e.g., 100% confluence), they are collected and passaged again in a new first keratinocyte culture medium at a predetermined ratio for 1 day to obtain second pluripotent stem cells. Therefore, culturing first pluripotent stem cells in a first keratinocyte culture medium, and then passaged again to the same medium after reaching a high density, ensures that the first pluripotent stem cells remain undifferentiated and possess good clonogenic ability, reducing the risk of spontaneous differentiation and improving batch-to-batch consistency.

[0069] Based on the above embodiments, as an optional implementation, the preset ratio is 1:2 to 1:6. Therefore, passage at this ratio can maintain the undifferentiated state of the first pluripotent stem cells and ensure their stable proliferation. Preferably, the preset ratio is 1:4.

[0070] Step S140: The second pluripotent stem cells are cultured in the second keratinocyte culture medium to obtain keratinocytes.

[0071] Specifically, second pluripotent stem cells are cultured in a second keratinocyte culture medium for 5 to 10 days to obtain keratinocytes. Both the first and second keratinocyte culture media contain calcium ions, with a higher calcium ion concentration in the first medium than in the second. Thus, by promoting pluripotent stem cell proliferation under higher calcium ion conditions and maintaining stemness and promoting maturation under lower calcium ion conditions, rapid and large-scale keratinocyte preparation can be achieved.

[0072] Based on the above embodiments, as an optional implementation, the concentration of calcium ions in the first keratinocyte culture medium is 1 mmol / L, and the concentration of calcium ions in the second keratinocyte culture medium is 0.01 mmol / L to 0.1 mmol / L. Preferably, the concentration of calcium ions in the first keratinocyte culture medium is 1 mmol / L, and the concentration of calcium ions in the second keratinocyte culture medium is 0.06 mmol / L. Therefore, a calcium ion concentration in the first keratinocyte culture medium within the above range is beneficial for further promoting the proliferation of pluripotent stem cells, and a calcium ion concentration in the second keratinocyte culture medium within the above range is beneficial for further maintaining the stemness of pluripotent stem cells and promoting their maturation, thereby further shortening the differentiation cycle of pluripotent stem cells and increasing the yield of pluripotent stem cells.

[0073] In this embodiment, the first and second keratinocyte culture media contain calcium ions as well as other basic components, such as basal culture medium E6, and basal culture medium E6 supplemented with 0.4 μg / mL Hydrocortison, 10 ng / mL EGF, 10 μmol / L Y27632, and 0.1% BSA (bovine serum albumin). Of course, those skilled in the art can also directly choose commercially available keratinocyte culture medium as the basal culture medium and add appropriate concentrations of calcium ions to it.

[0074] Based on the above embodiments, as a preferred embodiment, pretreated pluripotent stem cells are cultured in an induction medium for 7 days to obtain first pluripotent stem cells. On day 8, the first pluripotent stem cells are passaged into a first keratinocyte culture medium at a predetermined ratio and cultured for 7 days. After the confluence of the first pluripotent stem cells reaches 100%, the first pluripotent stem cells are collected. On day 15, they are further passaged into a new first keratinocyte culture medium at a predetermined ratio and cultured for 1 day to obtain second pluripotent stem cells. On day 16, the second pluripotent stem cells are placed in a second keratinocyte culture medium and cultured for 5 days to obtain keratinocytes. Thus, this embodiment can achieve large-scale (proliferation exceeding 400-fold) preparation of keratinocytes in only 21 days. Compared with the prior art, which usually requires more than 30 days to prepare keratinocytes, this embodiment shortens the differentiation cycle by about 30%, which not only significantly improves the differentiation efficiency but also significantly increases the cell yield.

[0075] The method for preparing keratinocytes derived from pluripotent stem cells provided in this embodiment pretreats pluripotent stem cells with multiple inhibitors before inducing differentiation with BMP4 and retinoic acid compounds. This inhibits the expression of the trophoblast characteristic gene CGB, thus blocking trophoblast lineage differentiation at its source before BMP4 and retinoic acid treatment. After the pluripotent stem cells are directionally induced to differentiate by BMP4 and retinoic acid compounds, a stepwise calcium ion concentration regulation strategy combined with continuous passage culture is used. First, culturing in a keratinocyte culture medium with a high calcium ion content promotes cell proliferation, and then culturing in a keratinocyte culture medium with a low calcium ion content maintains cell stemness and promotes maturation. This enables rapid and large-scale preparation of keratinocytes. Compared with existing technologies, this embodiment can shorten the differentiation cycle by approximately 30% and significantly increase cell yield. Furthermore, the preparation method provided in this embodiment is applicable to different types of pluripotent stem cell lines and has good stability and reproducibility.

[0076] The second aspect of this embodiment provides a 3D full-thickness skin model comprising keratinocytes prepared by the preparation method described in the first aspect.

[0077] The 3D full-thickness skin model was formed by seeding the keratinocytes onto the surface of a dermal substitute and then culturing them at an air-liquid interface.

[0078] Specifically, the 3D full-thickness skin model was prepared using the following method:

[0079] Offer genuine leather alternatives;

[0080] The keratinocytes described in the first aspect are seeded onto the surface of a dermal substitute, and then cultured at the air-liquid interface for 10 to 21 days to form a layered epidermal structure.

[0081] The keratinocytes obtained in this embodiment possess complete terminal differentiation potential. Under high calcium (1.8 mmol / L) induction, the expression of differentiation markers such as KRT1, KRT10, IVL, and FLG was significantly upregulated. When these keratinocytes were applied to a 3D full-thickness skin model and cultured at the air-liquid interface, HE staining confirmed that the keratinocytes could form a layered epidermal structure. Immunofluorescence confirmed that the basal cell layer expressed KRT5 / KRT14, the intermediate layer expressed KRT10, and the outermost layer expressed FLG. Its histological characteristics and molecular expression patterns were highly similar to those of natural human epidermis.

[0082] In this embodiment, the seeding density of keratinocytes on the surface of the dermal substitute is 2 × 10⁻⁶ per square centimeter. 4 Up to 1×10 6Each cell. Therefore, with the keratinocyte seeding density within the above range, a continuous basal cell layer can be ensured on the surface of the dermal substitute. Preferably, the keratinocyte seeding density on the surface of the dermal substitute is 5 × 10⁶ cells. 5 1 cell / Transwell (approximately 4.2 cm²).

[0083] Based on the above embodiments, as a preferred embodiment, after keratinocytes are seeded onto the surface of the dermal substitute, they are cultured at the gas-liquid interface for 14 days.

[0084] Based on the above embodiments, as an optional implementation, the 3D full-thickness skin model further includes a dermal scaffold and fibroblasts; wherein, the dermal scaffold is selected from any one or more combinations of collagen scaffolds, decellularized dermal scaffolds, and synthetic polymer scaffolds; the fibroblasts are selected from primary dermal fibroblasts, mesenchymal stem cells, or fibroblasts differentiated from mesenchymal stem cells. In this embodiment, the dermal substitute is formed by naturally solidifying a mixture of collagen, fibroblasts, and relevant culture medium. This embodiment does not further limit the specific preparation method of the dermal substitute; those skilled in the art can refer to existing patents or literature to prepare the dermal substitute.

[0085] The 3D full-thickness skin model provided in this embodiment involves seeding keratinocytes onto the surface of a dermal substitute and culturing them at the gas-liquid interface. This simulates the in vivo gas-liquid interface environment of skin tissue, promoting keratinocyte differentiation and improving skin barrier function, thereby constructing a structurally stable 3D full-thickness skin model. The 3D full-thickness skin constructed in this embodiment can not only be used in clinical applications for wound repair such as burns, but also serve as a standardized in vitro model in cosmetics and drug development. Compared with traditional animal experiments and primary cell models, the 3D full-thickness skin model provided in this embodiment has the following advantages:

[0086] (1) Reproducibility and standardization: The 3D full-thickness skin model provided in this embodiment uses pluripotent stem cells as the starting cells, which can establish a standardized cell bank with small batch-to-batch differences. This solves the problem of large differences and limited sources of primary cell donors, and provides a stable and reliable testing platform for cosmetic efficacy evaluation and drug screening.

[0087] (2) Significantly promotes wound repair: The 3D full-thickness skin model provided in this embodiment is constructed using keratinocytes. These keratinocytes have good wound repair capabilities. When this 3D skin model is used for wound repair such as burns, the regenerative capacity of keratinocytes can be used to effectively promote wound re-epithelialization and significantly improve the quality of skin healing.

[0088] (3) In line with the EU’s ban on animal testing: The 3D full-thickness skin model provided in this embodiment fully complies with the relevant provisions of the EU Cosmetics Regulation prohibiting animal testing. The 3D full-thickness skin model is constructed using keratinocytes and can reproduce the physiological structure of natural skin. It not only meets the regulatory requirements for safety testing of skin irritation, corrosiveness, phototoxicity, etc., but also has extremely high industrialization and promotion prospects.

[0089] (4) Complete skin barrier function: The 3D full-layer skin model provided in this embodiment has a complete layered epidermal structure and stratum corneum, which can simulate the complete skin epidermal structure and its barrier function;

[0090] (5) Scalability and customization: Due to the gene-editable characteristics of pluripotent stem cells, this embodiment can also construct specific hereditary skin disease models (such as ichthyosis and epidermolysis bullosa) through gene modification for screening and mechanism research of rare skin disease drugs, providing a new in vitro research platform for precision medicine.

[0091] The third aspect of this embodiment provides an application of keratinocytes as described in the first aspect, or a 3D full-thickness skin model as described in the second aspect, in at least one of the following:

[0092] To prepare drugs or medical devices for treating skin injuries;

[0093] To prepare an in vitro model for evaluating the efficacy of cosmetics;

[0094] Prepare in vitro models for drug screening.

[0095] Among them, skin trauma is preferably burns, diabetic foot ulcers, venous ulcers, or hereditary skin diseases; cosmetic efficacy evaluation includes skin irritation testing, skin corrosion testing, skin phototoxicity testing, skin barrier function evaluation, anti-aging efficacy evaluation, whitening efficacy evaluation, or moisturizing efficacy evaluation; drug screening includes skin drug permeability testing and skin drug metabolism.

[0096] The keratinocyte or 3D full-thickness skin model provided in this embodiment is formed by inducing differentiation of human pluripotent stem cells into keratinocytes, which are closer to real human tissue. Applying it to skin trauma, cosmetic efficacy evaluation and drug screening can not only significantly improve the physiological relevance and reliability of experimental results, but also establish an in vitro detection system with standardized and large-scale potential, which has significant clinical application value and broad industrial development prospects.

[0097] To provide a more detailed description of the present invention, specific embodiments will be used to further illustrate the invention. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods; unless otherwise specified, the materials and reagents used in the embodiments of the present invention are commercially available.

[0098] Example 1

[0099] In this embodiment, human embryonic stem cell line H1 (hESC H1) is used as a pluripotent stem cell. Keratinocytes are prepared using hESC H1, and the specific preparation method includes the following steps:

[0100] (1) Pretreatment: hESC H1 cells cultured on Matrigel-coated plates were passaged with EDTA solution and then seeded into new Matrigel-coated plates. After cell attachment, the medium was replaced with E8 medium containing a triple inhibitor combination for 24 hours of pretreatment. The composition and concentration of the triple inhibitor combination were as follows:

[0101] The SMAD2 / 3 pathway inhibitor is SB431542, and its concentration is 10 μmol / L.

[0102] The SMAD1 / 5 / 9 pathway inhibitor is LDN193189, used at a concentration of 100 nmol / L;

[0103] The FGF2 pathway inhibitor is PD0325901, used at a concentration of 100 nmol / L.

[0104] (2) BMP4 / RA directed differentiation

[0105] After pretreatment, the old culture medium was discarded to obtain pretreated pluripotent stem cells. These cells were then transferred to an induction medium containing inducing factors and cultured for 7 days, with the medium being replaced with fresh medium every 2 days. This stage is recorded as day 0 to day 7 of differentiation. The composition of the induction medium is as follows:

[0106] Basic culture medium: E6 medium;

[0107] BMP4: 20 ng / ml;

[0108] Retinic acid (RA): 1 μmol / L.

[0109] (3) Calcium ion regulation and continuous passage amplification

[0110] On day 7, the induction medium was discarded, and the cells were digested with TrypLE digestion solution for 5-10 minutes. After cell detachment, digestion was stopped using first keratinocyte culture medium (calcium ion concentration of 1 mmol / L), and the cells were collected to obtain first pluripotent stem cells. The first pluripotent stem cells were passaged into new culture dishes at a ratio of 1:4 and cultured in first keratinocyte culture medium for 7 days (i.e., day 8 to day 15 of differentiation). After the first pluripotent stem cells reached high density from day 8 to day 15, they were collected and passaged again at a ratio of 1:4 on day 15, and cultured in first keratinocyte culture medium for 1 day.

[0111] (4) Keratinocyte maturation culture

[0112] On day 16, the first keratinocyte culture medium was discarded and replaced with the second keratinocyte culture medium (calcium ion concentration of 0.06 mmol / L) for 5 more days (i.e., day 16 to day 21 of differentiation). After the culture was completed, the cells were collected, which are the high-purity and functional keratinocytes (iKERA) described in this example.

[0113] Cell counting was performed, from approximately 1 × 10⁻⁶ 6 Starting from a single hESC H1 cell, after 21 days of differentiation, more than 4 × 10⁶ cells can be obtained. 8 Each keratinocyte can proliferate more than 400 times, enabling rapid and large-scale keratinocyte preparation in this embodiment.

[0114] Example 2

[0115] In this embodiment, human induced pluripotent stem cell line ND1 (hiPSC ND1) is used as the pluripotent stem cell. Keratinocytes are prepared using hiPSC ND1. The specific preparation method is the same as that in Example 1, except that the type of pluripotent stem cell is different.

[0116] Example 3

[0117] In this embodiment, human embryonic stem cell line H9 (hESC H9) is used as a pluripotent stem cell. Keratinocytes are prepared using hESC H9. The specific preparation method is the same as that in Example 1, except that the type of pluripotent stem cell is different.

[0118] On day 21 of differentiation, the pluripotent stem cells obtained in Examples 2 and 3 were collected for qPCR detection and immunofluorescence staining, and the results are as follows. Figure 2 As shown.

[0119] Figure 2Figures show the verification results of the preparation method provided in this embodiment applied to different cell lines. Figures A and C show the qPCR detection results in different cell lines; Figures B and D show the immunofluorescence staining results in different cell lines. Figures A and C show that in both hiPSC H9 (A) and hESC ND1 (C) cell lines, the differentiated cells highly expressed the keratinocyte-specific markers KRT5, KRT14, and TP63. This indicates that pluripotent stem cells derived from hiPSC ND1 and hESC H9 have highly consistent marker expression patterns, with significantly upregulated mRNA expression levels of KRT5, KRT14, and TP63. Figures B and D show that KRT5 and KRT14 were co-expressed in both cell lines. These results demonstrate that the pluripotent stem cell preparation method provided in this embodiment is stable and efficient, applicable to different types of human pluripotent stem cell lines, and possesses good universality and reproducibility.

[0120] Example 4

[0121] This embodiment provides a 3D full-thickness skin model, which is constructed using the keratinocytes obtained in Example 1, and specifically includes the following steps:

[0122] (1) Preparation of dermal substitutes: Collagen solution (such as rat tail type I collagen) was mixed with concentrated DMEM medium, fetal bovine serum, and NaHCO3, and the pH was adjusted to neutral. The mixture was injected into Transwell chambers and human primary dermal fibroblasts were uniformly mixed in to achieve a final collagen concentration of 2 mg / ml and a fibroblast density of 2 × 10⁻⁶ cells / ml. 5 Cells / ml. After the collagen gel solidifies, fibroblast culture medium is added on top of the gel and cultured for 1-2 days to allow the dermal substitute to shrink and stabilize, thus obtaining the dermal substitute.

[0123] (2) Inoculation of keratinocytes: The iKERA cells prepared in Example 1 were digested, counted, and resuspended in a high-calcium keratinocyte culture medium (calcium ion concentration of 1.8 mmol / L). The cell suspension was inoculated onto the surface of the dermal substitute prepared in Step 1 at an inoculation density of 5 × 10⁻⁶ cells / day. 5 1 cell / Transwell (approximately 4.2 cm²).

[0124] (3) Gas-liquid interface culture: After the cells adhered, the culture medium above the Transwell chamber was aspirated, exposing the epidermal cell layer to the air. Culture medium (a mixture of concentrated DMEM, L-glutamin, fetal bovine serum, gentamycin, and NaHCO3) was added only to the bottom of the chamber, and gas-liquid interface culture was performed at 37°C. The culture time was 14 days, during which the culture medium at the bottom was replaced every 2-3 days. After the gas-liquid interface culture was completed, a 3D full-thickness skin model with a layered epidermal structure was obtained.

[0125] Figure 3 Figure 1 shows the functional verification diagrams of the keratinocytes (iKERA) prepared in this embodiment. Figure A shows the changes in the content of terminal differentiation markers in iKERA after induction; Figure B shows the HE staining results of the layered epidermal structure; Figures C and D show the immunofluorescence staining results of the layered epidermal structure, respectively. Figure A shows that after high calcium (calcium ion concentration of 1.8 mmol / L) induction, the mRNA expression levels of terminal differentiation markers KRT1, KRT10, IVL, and FLG in iKERA were significantly upregulated. Figure B shows that after iKERA was seeded onto the surface of a dermal substitute and cultured at the gas-liquid interface, HE staining results showed that the cells formed a layered epidermal structure highly similar to natural skin. Figures C and D show that this 3D full-thickness skin model has a clear layered structure, with the basal cell layer expressing KRT5 / KRT14 / TP63, the middle layer expressing KRT10, and the outermost layer expressing FLG, confirming its complete barrier function.

[0126] Experimental Example 1

[0127] This experimental example is used to verify the ability of the keratinocytes provided in this embodiment to repair burn wounds. The specific verification method includes the following steps:

[0128] (1) Animal model establishment: After anesthetizing immunodeficient mice (such as nude mice), a full-thickness circular skin defect with a diameter of 8 mm was created on both sides of the midline of their backs using a skin biopsy device to establish an acute trauma model.

[0129] (2) Cell transplantation: Experimental animals were randomly divided into two groups: a control group (injected with PBS buffer) and an experimental group (injected with a suspension containing iKERA cells prepared in Example 1). On day 0 after trauma initiation, 1×10 6 Multiple iKERA cells (resuspended in 100 μl PBS) were injected subcutaneously at multiple points around the wound edge.

[0130] (3) Wound healing assessment: The wound was photographed on days 0, 3, 7, 10 and 14 after the injury, and the wound healing rate was calculated (healing rate = (initial wound area - wound area on the day of measurement) / initial wound area × 100%).

[0131] Figure 4 Figures show the in vivo functional validation of the keratinocytes (iKERA) prepared in this embodiment for wound repair. Figures A and B are representative wound photographs of a full-thickness skin defect model in immunodeficient mice at different time points; Figure C is a statistical analysis of wound healing rate; Figures D and E are statistical graphs of wound skin sections and epidermal thickness, respectively. Figures A and B show that, compared with the control group (Mock, without iKERA cells), the wounds in the iKERA-treated group healed faster at 10 and 14 days. Figure C shows that, from day 10, the wound healing rate in the iKERA-treated group was significantly higher than that in the control group (*p < 0.05), indicating that iKERA can significantly promote wound repair. Figure D shows that, compared with the control group (Mock), the iKERA-treated wounds had a thicker epidermal structure. These results indicate that, starting from day 10 post-traumatic injury, the wound healing rate in the experimental group (iKERA-treated group) was significantly faster than that in the control group. By day 14, the wounds in the experimental group were almost completely closed, while the control group still had large unhealed wounds. These results fully demonstrate that the keratinocytes prepared in this invention have excellent wound repair capabilities in vivo and can significantly promote wound healing.

[0132] Experimental Example 2

[0133] This experimental example is used to verify the advantages of the preparation method provided in this embodiment for preparing keratinocytes compared with traditional preparation methods.

[0134] Compared to the preparation method in Example 1, the conventional preparation method for keratinocytes lacks steps (1) and (3). Specifically, after culturing pluripotent stem cells in an induction medium containing inducing factors for 7 days, the cells are then transferred to a second keratinocyte culture medium (calcium ion concentration of 0.06 mmol / L) for further culture. 3i+BMP4 / RA represents keratinocytes prepared using the method of this example, while BMP4 / RA represents keratinocytes prepared using the conventional method. The resulting cells are as follows: Figure 5 and Figure 6 The diagram shown is a structural schematic.

[0135] Figure 5This diagram illustrates the phylogenetic differentiation pathway of the traditional preparation method. Figures A and B show the phylogenetic differentiation induced by the traditional preparation method, respectively. Figures C through E show the phylogenetic differentiation induced by the traditional preparation method, analyzed using RNA-seq. Figures A through E demonstrate that the traditional preparation method induces phylogenetic differentiation and expression of the trophoblast characteristic genes CGB and GCM1.

[0136] Figure 6 This diagram illustrates the comparison of lineage differentiation pathways between the traditional preparation method and the method described in this embodiment. Figure A shows the trophoblast lineage differentiation induced by BMP4 / RA inhibition using dual SMAD2 / 3 and SMAD1 / 5 / 9 pathways; Figure B shows the trophoblast and epidermal ectoderm lineage differentiation after the addition of triple inhibitors; Figures C to D show the results of immunofluorescence and flow cytometry analysis confirming that the addition of triple inhibitors (3i) suppressed the trophoblast lineage while maintaining the expression of epidermal ectoderm characteristic markers. Figures E to G show the results of RNA-seq analysis after 3i optimization, which suppressed trophoblast lineage genes while maintaining the expression of epidermal ectoderm characteristic genes. Figure H illustrates the lineage differentiation induced by the traditional BMP4 / RA method and the optimized 3i+BMP4 / RA method.

[0137] As shown in Figure A, the simultaneous addition of SB431542 and LDN193189 inhibited SMAD2 / 3 and SMAD1 / 5 / 9, suppressing the expression of the trophoblast marker gene CGB while maintaining the expression of the epidermal ectoderm marker gene TP63. Figure B shows that adding the FGF2 pathway inhibitor PD0325901 to the dual SMAD2 / 3 and SMAD1 / 5 / 9 pathway inhibition further promoted epidermal ectoderm differentiation, thus significantly promoting epidermal ectoderm differentiation. Figures C through G demonstrate that pretreatment with triple inhibitors significantly suppressed trophoblast lineage deviation during differentiation using traditional methods.

[0138] Figure 7This diagram illustrates the process of keratinocyte iKERA differentiation in this embodiment. Figure A shows the cell morphology of keratinocytes at various stages of differentiation; Figure B shows the proliferation curve of keratinocytes during differentiation; Figure C shows the gene expression curves during differentiation using the method of this embodiment and traditional methods; Figures D to E show the immunofluorescence analysis on day 21 of differentiation using the method of this embodiment and traditional methods. Figure A details the complete process from pluripotent stem cells, through four stages: "triple inhibition pretreatment," "BMP4 / RA directed differentiation," "calcium-regulated amplification and continuous passage," and "keratinocyte maturation culture (low calcium)," ultimately obtaining functional keratinocytes (iKERA) within 21 days. Figure B shows that after 21 days of differentiation, cell proliferation exceeds 400-fold, indicating that the preparation method of this embodiment is suitable for large-scale cell production. Figure C shows that the preparation method of this embodiment can achieve significantly increased expression of keratinocyte characteristic genes KRT5 and KRT14 at day 21 of differentiation. As can be seen from Figures D and E, the preparation method of this embodiment has significantly stronger expression of TP63, KRT5, and KRT14 compared with the traditional preparation method.

[0139] Experimental Example 3

[0140] This experimental example is used to verify the effects of passage treatment and calcium ion concentration on the preparation of keratinocytes by the preparation method provided in this embodiment.

[0141] The pluripotent stem cells pretreated with the triple inhibitor in step (1) of Example 1 and the first pluripotent stem cells obtained after induction with BMP4 and RA in step (2) were passaged or not passaged on day 8, respectively, and then cultured in the first keratinocyte culture medium (calcium ion concentration of 1 mmol / L) until day 15.

[0142] Figure 8 This diagram illustrates the different treatment methods used to promote keratinocyte differentiation. Figure A shows the experimental procedure and representative cell morphology images, with a scale bar of 100 μm used when capturing cell morphology images. Figure B shows the fold change in cell proliferation from day 8 to day 15 under different treatment methods. Figure C shows the qPCR analysis results of keratinocyte marker genes (TP63, KRT5, KRT14) on day 15 under different treatment methods (n=4, *p < 0.05). Figure 2 It can be seen that performing passage treatment on day 8 in this embodiment is beneficial to further increase the cell proliferation rate.

[0143] The pluripotent stem cells pretreated with the triple inhibitor in step (1) of Example 1 and the first pluripotent stem cells obtained after induction with BMP4 and RA in step (2) were passaged on day 8, and then cultured in the first keratinocyte culture medium (calcium ion concentration of 1 mmol / L) and the second keratinocyte culture medium (calcium ion concentration of 0.06 mmol / L) until day 15.

[0144] Subsequently, the cells were passaged again on day 15 and cultured in first keratinocyte culture medium (calcium ion concentration of 1 mmol / L) and second keratinocyte culture medium (calcium ion concentration of 0.06 mmol / L) until day 21. The first pluripotent stem cells cultured in the first keratinocyte culture medium were always cultured in the same medium, and the first pluripotent stem cells cultured in the second keratinocyte culture medium were always cultured in the same medium.

[0145] Figure 9 This diagram illustrates the phased calcium regulation that promotes keratinocyte differentiation. Figure A shows the experimental procedure and representative cell morphology images from day 8 to day 15, with a scale bar of 100 μm when the cell morphology images were taken. Figure B shows the change in cell proliferation fold from day 8 to day 15 under different calcium ion concentrations. Figure C shows the qPCR analysis results (F) of keratinocyte marker genes (TP63, KRT5, KRT14) at day 15 under different calcium ion concentrations. Figure D shows the experimental procedure and representative cell morphology images from day 15 to day 21, with a scale bar of 100 μm when the cell morphology images were taken. Figure E shows the change in cell proliferation fold from day 15 to day 21 under different calcium ion concentrations. Figure F shows the qPCR analysis results (I) of keratinocyte marker genes (TP63, KRT5, KRT14) at day 21 under different calcium ion concentrations (n=4, *p < 0.05, ns indicates no significant difference).

[0146] As shown in Figures A through C, during days 8-15, 1 mmol / L The cell proliferation rate of the group was significantly faster than that of 0.06 mmol / L. Group, indicating that during days 8 to 15, select a solution containing 1 mmol / L. The culture medium was better; as shown in Figures D to F, during days 15-21, 1 mmol / L The cell proliferation rate of the group was significantly slower than that of 0.06 mmol / L mM. Group 1, indicating that the patient selected a 0.06 mmol / L solution during days 16 to 21. The culture medium was better. Furthermore, since the cells reached high density on day 15 and were passaged again on day 15, the previous culture medium of 1 mmol / L was continued on day 15. The culture medium is better.

Claims

1. A method for preparing keratinocytes derived from pluripotent stem cells, characterized in that, Includes the following steps: Pluripotent stem cells are passaged, and the passaged pluripotent stem cells are pretreated with multiple inhibitors. After the pretreatment, pretreated pluripotent stem cells are obtained. The multiple inhibitors include SMAD2 / 3 pathway inhibitors, SMAD1 / 5 / 9 pathway inhibitors and FGF2 pathway inhibitors. The pretreated pluripotent stem cells were cultured in an induction medium to obtain first pluripotent stem cells, wherein the induction medium contained BMP4 and retinoic acid compounds. The first pluripotent stem cells were passaged into a first keratinocyte culture medium at a preset ratio and cultured. After the first pluripotent stem cells reached a high density, the first pluripotent stem cells were collected and passaged into a first keratinocyte culture medium at a preset ratio to obtain second pluripotent stem cells. The second pluripotent stem cells were cultured in a second keratinocyte culture medium to obtain keratinocytes; Both the first keratinocyte culture medium and the second keratinocyte culture medium contain calcium ions, with the calcium ion concentration in the first keratinocyte culture medium being higher than that in the second keratinocyte culture medium.

2. The method for preparing keratinocytes derived from pluripotent stem cells according to claim 1, characterized in that, The multiple inhibitors pretreat the passaged pluripotent stem cells for 12 to 48 hours. The SMAD2 / 3 pathway inhibitor is selected from at least one of SB431542, A8301 and pharmaceutically acceptable salts thereof, and the concentration of the SMAD2 / 3 pathway inhibitor used is from 1 μmol / L to 20 μmol / L. The SMAD1 / 5 / 9 pathway inhibitor is selected from at least one of LDN193189 and its pharmaceutically acceptable salts, and the concentration of the SMAD1 / 5 / 9 pathway inhibitor used is from 10 nmol / L to 1000 nmol / L; The FGF2 pathway inhibitor is selected from at least one of PD0325901, PD173074, SU5402 and their pharmaceutically acceptable salts, and the concentration of the FGF2 pathway inhibitor used is from 10 nmol / L to 1000 nmol / L.

3. The method for preparing keratinocytes derived from pluripotent stem cells according to claim 1, characterized in that, The concentration of BMP4 in the induction medium is from 1 ng / mL to 100 ng / mL, and the concentration of the retinoic acid compound is from 0.1 μmol / L to 10 μmol / L. The retinoic acid compound is selected from at least one of retinoic acid, TTNPB, and pharmaceutically acceptable salts thereof.

4. The method for preparing keratinocytes derived from pluripotent stem cells according to claim 1, characterized in that, Pretreated pluripotent stem cells were cultured in induction medium for 5 to 9 days, with fresh induction medium replaced every 2 days.

5. The method for preparing keratinocytes derived from pluripotent stem cells according to claim 1, characterized in that, The first pluripotent stem cells are passaged into the first keratinocyte culture medium at a preset ratio and cultured for 5 to 10 days. The first pluripotent stem cells are then collected and passaged into the first keratinocyte culture medium at a preset ratio and cultured for 1 day. The preset ratio is 1:2 to 1:6, and the concentration of calcium ions in the first keratinocyte culture medium is 1 mmol / L.

6. The method for preparing keratinocytes derived from pluripotent stem cells according to claim 1, characterized in that, The second pluripotent stem cells were cultured in a second keratinocyte culture medium for 5 to 10 days; The concentration of calcium ions in the second keratinocyte culture medium was 0.01 mmol / L to 0.1 mmol / L.

7. A 3D full-thickness skin model, characterized in that, The 3D full-thickness skin model includes keratinocytes prepared by the preparation method according to any one of claims 1 to 6.

8. The 3D full-thickness skin model according to claim 7, characterized in that, The 3D full-thickness skin model was formed by seeding the keratinocytes onto the surface of a dermal substitute and then culturing them at an air-liquid interface. The seeding density of the keratinocytes was 2 × 10⁻⁶ per square centimeter. 4 Up to 1×10 6 Each cell.

9. The 3D full-thickness skin model according to claim 7, characterized in that, The 3D full-thickness skin model shown also includes a dermal scaffold and fibroblasts; The dermal scaffold is selected from any one or more combinations of collagen scaffolds, decellularized dermal scaffolds, and synthetic polymer scaffolds; the fibroblasts are selected from primary dermal fibroblasts, mesenchymal stem cells, or fibroblasts differentiated from mesenchymal stem cells.

10. The keratinocytes prepared by the method according to any one of claims 1 to 6, or the 3D full-thickness skin model according to any one of claims 7 to 9, in at least one of the following: To prepare drugs or medical devices for treating skin injuries; To prepare an in vitro model for evaluating the efficacy of cosmetics; Prepare in vitro models for drug screening.

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