3D cell spheroids for treating skin defects and preparation method and application thereof

Abstract

This invention discloses a 3D cell sphere for treating skin defects, its preparation method, and its applications, belonging to the field of biomedical materials. The 3D cell sphere is formed by the self-assembly of keratinocytes (KCs) and cells capable of secreting extracellular matrix, possessing a biomimetic hierarchical structure similar to natural skin, filled with extracellular matrix, and the KCs can form functional epidermal-associated structures. The preparation method involves obtaining EKCs through directed induction differentiation of pluripotent stem cells, which are then spontaneously assembled with cells capable of secreting extracellular matrix to form 3D cell spheres. The 3D cell sphere exhibits excellent biocompatibility, a simple preparation process, and can be mass-produced, with convenient operation. It is suitable for repairing various acute and chronic skin defects such as large-area thermal burns, diabetic foot ulcers, and pressure ulcers, effectively accelerating wound healing and improving the quality of epidermal regeneration. It addresses the pain points of traditional treatments, such as scarcity of skin cells, complex procedures, and poor efficacy, providing a safe and efficient novel cell therapy for skin defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a 3D cell sphere for treating skin defects, its preparation method, and its application. Background Technology

[0002] Extensive thermal burns, diabetic foot ulcers, and pressure ulcers, among other acute and chronic skin defects, have become a global medical and economic burden, affecting over 100 million people annually. Timely and effective wound repair is crucial for saving lives and improving prognosis. Currently, autologous skin grafting remains the gold standard for treating extensive injuries. However, this standard treatment method has limitations such as a scarcity of autologous skin donors, secondary damage to the donor site, immune rejection, and scar hyperplasia and contracture. This leads to severe functional impairment and psychological burden on patients, resulting in a sharp decline in their quality of life. Furthermore, the supply-demand imbalance between donor and graft sites becomes increasingly pronounced as the wound area increases. Although allogeneic skin or autologous epidermal cell culture transplantation is used clinically, it remains constrained by insufficient sources and long culture times.

[0003] Against this backdrop, cell therapy developed based on the proliferation and differentiation capabilities of stem cells has brought a revolutionary glimmer of hope to this challenge. Its core advantage lies in its ability to start from the source of seed cells, constructing viable skin tissue in vitro and implanting it into the wound, thus achieving true "regeneration" rather than just "repair." This strategy not only fundamentally solves the problem of skin cell shortage but also regulates the wound microenvironment by secreting various growth factors, promoting graft-host integration and potentially ultimately achieving scarless healing and reconstruction of skin appendages. Human embryonic stem cells (hESCs), as pluripotent stem cells, exhibit unparalleled potential and advantages due to their proliferation and differentiation capabilities, making them an excellent source of skin seed cells. First, hESCs possess near-unlimited self-renewal capacity, enabling large-scale expansion in vitro, providing an inexhaustible cell bank for the industrial production of epidermal grafts. Secondly, and more importantly, by precisely manipulating signaling pathways such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP), hESCs can be efficiently induced to differentiate into core skin components such as keratinocytes (KCs) and melanocytes. Recent studies have further demonstrated that skin organoids constructed using hESCs or induced pluripotent stem cells can reproduce complex multilayered epidermal structures, even including functional appendages such as hair follicles, sebaceous glands, and sweat glands, achieving a fundamental leap from "wound repair" to "tissue regeneration."

[0004] However, the clinical translational efficacy of seed cells largely depends on their transplantation method. The most common method, cell suspension transplantation, is too prone to loss, making it difficult to control transplantation efficiency. Furthermore, cells entering the wound die in large numbers due to changes in osmotic pressure and environment after leaving the culture medium. While cell sheet technology can preserve the extracellular matrix and improve cell survival, it requires specific culture conditions, is time-consuming, has brittle mechanical properties, and is difficult to operate surgically. Current bioscaffold materials provide physical support, but their degradation rate is difficult to synchronize with tissue integration, and some natural matrices (such as Matrigel) pose heterogeneity risks and batch-to-batch variations. Therefore, tissue-engineered skin often achieves its therapeutic effect through the paracrine effects of cells within the scaffold. Studies have found that 3D cell clumps or cell spheres, by mimicking the in vivo microenvironment (Niche), can significantly enhance intercellular interactions (juxtacrine) and paracrine effects (paracrine). In the natural physiological structure of skin, dermal fibroblasts provide crucial mechanical support and biochemical regulatory signals for the epidermis by secreting abundant collagen and ECM. Studies have also shown that grafts lacking mesenchymal-ectoderm signals (such as Gibbin-dependent signals) can lead to incomplete epidermal differentiation and maturation.

[0005] Therefore, developing a novel treatment method that can overcome the limitations of skin source, has excellent biocompatibility, and can rebuild the skin barrier in a short time has become an urgent goal in the field of regenerative medicine. Summary of the Invention

[0006] This invention discloses a 3D cell sphere for treating skin defects, its preparation method, and its applications. The 3D cell sphere is formed by the self-assembly of keratinocytes (KCs) and cells capable of secreting extracellular matrix, possessing a biomimetic hierarchical structure similar to natural skin. Its interior is filled with natural extracellular matrix secreted by HFBs, and the KCs can form functional epidermal-associated structures. The core of its preparation method is: obtaining functional EKCs through directed induction differentiation of pluripotent stem cells, then mixing them with cells capable of secreting extracellular matrix under specific conditions, followed by spontaneous assembly to form 3D cell spheres. This 3D cell sphere exhibits excellent biocompatibility, a simple preparation process, and can be mass-produced with ease. It can be used to prepare formulations for treating skin defects, suitable for repairing various acute and chronic skin defects such as large-area thermal burns, diabetic foot ulcers, and pressure ulcers. It can effectively accelerate wound healing and improve the quality of epidermal regeneration, solving the pain points of traditional treatments such as scarcity of skin cells, complex procedures, and poor efficacy, providing a safe and efficient new cell therapy solution for skin defect repair.

[0007] On one hand, the present invention provides a 3D cell sphere, which is formed by the self-assembly of keratinocytes and cells that can secrete extracellular matrix; the 3D cell sphere is surrounded by a keratinocyte layer on the outside and cells that can secrete extracellular matrix on the inside.

[0008] The core role of the cells capable of secreting extracellular matrix (ECM) in the 3D cell spheres is to secrete ECM (such as type I collagen) to construct an in-situ support network, providing physical support, a suitable microenvironment, and epithelial-mesenchymal interaction signals for keratinocytes (KCs). Essentially, this involves "achieving synergistic sphere formation and repair functions through ECM secretion." Based on this core function, the cells capable of secreting ECM encompass all cell types possessing this function, such as human fibroblasts (HFBs) and various mesenchymal stem cells. Although these cells originate from different sources, they can all secrete ECM components such as collagen and fibronectin, which self-assemble with KCs to form 3D cell spheres with a biomimetic hierarchical structure, effectively achieving the core effects of resisting wound impact, improving cell survival rate, and promoting epidermal regeneration.

[0009] Currently, the treatment of skin defects such as large-area burns and chronic ulcers mainly relies on autologous skin grafts, but this method suffers from problems such as scarcity of donor skin, secondary damage to the donor site, immune rejection, and scar hyperplasia. Pluripotent stem cell-induced keratinocytes (EKCs), derived from pluripotent stem cells (which can proliferate indefinitely), can be industrially produced on a large scale, perfectly solving the problem of scarcity of donor skin and serving as the seed cells for this invention. However, cells cultured in vitro have very strict requirements for the external environment; they die rapidly after leaving the liquid environment of the culture medium due to changes in osmotic pressure. Therefore, to successfully transplant EKCs into animals, a suitable microenvironment needs to be provided. In skin tissue, human fibroblasts (HFBs) in the dermis secrete a large amount of extracellular matrix, playing a crucial supporting role for epidermal cells and providing a suitable microenvironment for epidermal cell growth and migration. During the exploration process, the team discovered that pure EKCs are difficult to aggregate tightly in the absence of serum, but in the presence of another cell type that secretes extracellular matrix, the two cell types can spontaneously form a 3D spherical structure. Therefore, the invention team conceived of constructing 3D cell spheres by introducing cells capable of secreting extracellular matrix (ECM) and building an in situ ECM network using these cells to provide an extracellular matrix environment for EKCs, thus offering immediate physiological support and protection for in vivo transplantation of epidermal cells. This 3D synergistic model not only effectively resists the severe impact of the wound microenvironment and prevents cell death, but also promotes high-quality integration and multilayered development of the graft and host by mimicking epithelial-mesenchymal interactions (EMI) during embryonic development.

[0010] In existing technologies, other transplantation methods include single-cell suspension transplantation, cell suspension transplantation, constructing cell sheets, or using biomaterials to load EKCs. Cell suspension transplantation is prone to loss and has low cell viability; cell sheet technology is complex and time-consuming; and biomaterial-loaded EKCs have problems such as inconsistent degradation rates and the risk of xenogeneicity. Compared with these transplantation methods, the 3D cell spheroid structure provided by this invention has multiple advantages. First, the internal structure of the cell spheroid forms a tissue-like three-dimensional microstructure through tight junctions, gap junctions, and cell-matrix interactions. This significantly reduces the stress response and apoptosis rate of cells during separation, processing, and transplantation, which is conducive to the rapid adaptation and functional performance of transplanted cells. Second, its relatively large volume makes it less likely to be washed away by wound exudate or blood flow after transplantation, thereby improving the retention and retention efficiency at the injury site and ensuring continuous repair effects. Overall, cell sphere transplantation, by mimicking the spatial arrangement and interaction network of cells in natural skin tissue, has shown superior potential compared to dispersed cell transplantation in improving cell survival rate, maintaining cell functional status, enhancing paracrine effects, and potentially regulating the local immune microenvironment.

[0011] Furthermore, the total number of keratinocytes and cells capable of secreting extracellular matrix is ​​20,000 to 100,000.

[0012] The formation of 3D cell spheroids is mainly related to cells that secrete extracellular matrix; different numbers of fibroblasts result in cell spheroids of varying sizes. When the spheroid becomes too large, the central cells may undergo apoptosis due to insufficient nutrient supply.

[0013] In some methods, the ratio of EKCs to extracellular matrix-secreting cells (HFBs) was fixed, and the total number of both types of cells was varied to create 3D cell spheres. Live / dead cell staining was then performed. Results showed that cell spheres composed of 100,000 and 150,000 cells already contained a large number of dead cells by day 3, with the central cells having undergone apoptosis. Cell spheres composed of 50,000 cells had fewer dead cells, and the number of dead cells decreased further with decreasing cell count. Considering that the spheres were too small for subsequent operations, the final choice was to select 20,000 to 100,000 cells to self-assemble into 3D cell spheres.

[0014] Preferably, the sum of the number of keratinocytes and cells that can secrete extracellular matrix is ​​50,000.

[0015] Furthermore, the ratio of the number of keratinocytes to the number of cells that can secrete extracellular matrix is ​​1 to 3:1.

[0016] The ratio of EKCs to cells that secrete extracellular matrix is ​​directly related to the density of 3D cell spheres. The higher the proportion of cells that secrete extracellular matrix and the fewer EKCs, the more tightly packed the cells inside the cell sphere, and the more robust the sphere. Conversely, the lower the proportion of cells that secrete extracellular matrix and the more EKCs, the more gaps exist inside the cell sphere, making it difficult to form a solid spherical structure, and it is easy to break during the transfer process.

[0017] In some methods, experiments have verified that setting the ratio of EKCs to cells that secrete extracellular matrix (HFBs) below 3:1 (i.e., EKCs should not exceed three times the number of HFBs) allows HFBs to aggregate into relatively firm clusters inside the 3D cell spheres, while a continuous and relatively larger layer of EKCs cells forms on the outer layer of the 3D cell spheres.

[0018] Preferably, the ratio of keratinocytes to cells that secrete extracellular matrix is ​​3:1.

[0019] Furthermore, the cells capable of secreting extracellular matrix include any one of human fibroblasts, various types of mesenchymal stem cells, various types of endothelial cells, and chondrocytes.

[0020] Furthermore, the cells that secrete extracellular matrix are human fibroblasts.

[0021] Furthermore, the 3D cell spheres are filled with type I collagen secreted by human fibroblasts; the keratinocytes are induced to differentiate from pluripotent stem cells.

[0022] Type I collagen (COL I) is a core structural protein of the extracellular matrix (ECM), mainly secreted by HFBs. Its core function is to provide physical support and structural stability for 3D cell spheres, fill the gaps between EKCs and HFBs, enhance the mechanical stability of cell spheres, and provide a suitable microenvironment for cell attachment, proliferation, and differentiation. It is a key component in ensuring that 3D cell spheres resist wound impact and promote epidermal regeneration.

[0023] Furthermore, the keratinocytes can differentiate in vitro to form multilayered epidermal structures and desmosome structures.

[0024] The multilayered epidermal structure is formed by the orderly arrangement of multiple layers of epidermal cells (EKCs). Natural skin has a hierarchical relationship of "basal layer - spinous layer - granular layer - stratum corneum". The EKCs provided by this invention can be induced to differentiate into a multilayered epidermal structure in vitro and possess complete skin barrier function. Desmosomes are key connecting structures between epidermal cells, tightly connecting adjacent keratinocytes, enhancing intercellular adhesion, maintaining the structural stability of the multilayered epidermis, preventing cell shedding, and ensuring the integrity of the regenerated epidermis. The multilayered epidermal structures that the EKCs provided by this invention can differentiate into in vitro contain desmosome structures. Therefore, the EKCs provided by this invention are fully functional and can rapidly form a continuous epidermal barrier in vivo, avoiding the problem of "thin regenerated epidermis and weak barrier function" caused by incomplete function of traditional grafts.

[0025] On the other hand, the present invention provides a method for preparing the above-mentioned 3D cell spheres, wherein the keratinocytes are mixed with cells that can secrete extracellular matrix to spontaneously form 3D cell spheres.

[0026] Furthermore, the keratinocytes are directed to differentiate from pluripotent stem cells.

[0027] Compared to conventional autologous or allogeneic skin transplantation in clinical practice, EKC-based cell therapy strategies offer a significant advantage in scalability of sourcing. Pluripotent stem cells possess near-unlimited self-renewal capacity, allowing for large-scale induction into EKCs. These cells can then be cryopreserved long-term through cell banks, enabling rapid thawing and supply when urgently needed. Furthermore, the cell sphere preparation process established in this invention is simple and time-efficient. Spontaneous cell assembly requires no exogenous scaffolds or complex equipment, enabling the production of large quantities of standardized transplantable cell spheres within 24 hours, demonstrating good feasibility for clinical translation.

[0028] Furthermore, the pluripotent stem cells include human embryonic stem cells and induced pluripotent stem cells.

[0029] The human embryonic stem cells (hESCs) possess strong pluripotency and high induction efficiency, while the induced pluripotent stem cells (iPSCs) can be derived from the patient's own body, reducing the risk of immune rejection and adapting to diverse clinical needs. Both types of stem cells have unlimited proliferation and directed differentiation capabilities, allowing for the stable acquisition of high-purity, highly active EKCs. hESCs are suitable for the production of general-purpose cell preparations, while iPSCs are suitable for personalized treatment, covering a wider range of clinical scenarios.

[0030] Furthermore, the pluripotent stem cells are preferably hESCs.

[0031] Furthermore, the directed induction process employs a three-stage induction method; the first stage of induction induces pluripotent stem cells into epidermal lineage precursor cells; the second stage of induction induces epidermal lineage precursor cells into immature keratinocytes; and the third stage of induction induces immature keratinocytes into functional keratinocytes.

[0032] Furthermore, the culture medium used in the first induction stage includes DKSFM, the culture medium used in the second induction stage includes CNT 07, and the culture medium used in the third induction stage includes CNT 07.

[0033] The directed induction process of this invention adopts a three-stage design. In the first stage, a culture medium containing DKSFM is used to adapt pluripotent stem cells to early directed differentiation into the epidermal lineage. In the second and third stages, CNT07 culture medium is used to support cell proliferation and maintain stemness. The stage-specific culture medium precisely matches the physiological laws of EKC differentiation, which avoids the problems of unclear differentiation direction and low purity caused by traditional mixed culture media, reduces cell stress response, significantly improves induction efficiency and cell activity, and still has excellent proliferation capacity within 5 generations. This lays a key foundation for the large-scale and standardized preparation of 3D cell spheres.

[0034] In some methods, H9 hESCs are induced into EKCs in a three-stage process.

[0035] In some methods, the computational methods mentioned by Ruiz-Torres have shown that EKCs have good proliferation capabilities.

[0036] In some methods, EKCs induced by gas-liquid culture were differentiated and further matured. It was found that EKCs were relatively stable during the culture process and had the ability to construct multilayered epidermis in vitro. Moreover, the epidermal structure induced by EKCs finally conformed to the physiological structure of normal skin.

[0037] Furthermore, RA and BMP4 are added to the culture medium used in the first induction stage; EGF is added to the culture medium used in the second induction stage; and Y27632 is added to the culture medium used in the third induction stage.

[0038] In the three-stage induction process, this invention further precisely optimizes the factor combination of the culture medium: In the first stage, RA and BMP4 are added, whose synergistic effect efficiently promotes the directed differentiation of pluripotent stem cells into the epidermal lineage; in the second stage, EGF is added, which significantly enhances the proliferation capacity of keratinocytes, ensuring a sufficient number of seed cells to meet the needs of large-scale preparation; in the third stage, Y27632 is added, which effectively inhibits the ROCK pathway, reduces apoptosis, and increases cell adhesion rate, while maintaining the stemness and differentiation potential of keratinocytes. This staged and precise factor addition design avoids the problems of chaotic factor combinations and imprecise regulation in traditional induction protocols, achieving synergistic regulation of "directed differentiation, proliferation and expansion, and stemness maintenance," and ensuring excellent proliferative activity and functional integrity within 5 generations, providing a key guarantee for the stable preparation of 3D cell spheres.

[0039] In another aspect, the present invention provides the use of the 3D cell spheres as described above in the preparation of formulations that improve the therapeutic effect on skin defects.

[0040] In some methods, the therapeutic effects of the 3D cell sphere transplantation protocol provided by this invention and other existing transplantation protocols (including dripping EKCs cell suspension, constructing EKCs cell sheets, and loading EKCs cells onto different ECM materials) on mouse skin defects were compared. The results showed that the 3D cell sphere transplantation protocol comprehensively outperformed other transplantation protocols in terms of epidermal continuity, regeneration thickness, cell survival, and biocompatibility, making it a safer and more effective cell transplantation strategy for skin defect repair.

[0041] The present invention has the following beneficial effects: 1. The 3D cell sphere structure is biomimetic and stable, ensuring cell survival and function: The 3D cell sphere has a continuous EKCs layer on the outside and aggregated cells (HFBs) that can secrete extracellular matrix and secreted EMC (COL I) on the inside, perfectly mimicking the tissue structure and microenvironment of the natural skin "epidermis-dermis"; this structure can effectively resist changes in wound osmotic pressure and inflammatory impact, avoid cell loss and death, and improve the survival rate of EKC transplantation.

[0042] 2. In this invention, the total number of cells in the 3D cell spheres and the ratio of EKCs to HFBs are screened. The preferred total number of cells in the 3D cell spheres is 50,000, and the ratio of EKCs to HFBs is 3:1. This ensures that the 3D cell spheres aggregate into relatively solid clusters inside, while the outer layer forms a continuous layer of EKCs cells with a relatively larger number of cells.

[0043] 3. Significant therapeutic effect, achieving high-quality epidermal regeneration: After transplantation of 3D cell spheres, they can quickly establish themselves in the wound, forming continuous epidermis within 10 days post-surgery, and achieving 100% wound closure within 20 days.

[0044] 4. Convenient preparation and operation, and high feasibility for clinical translation: EKCs induction adopts a standardized three-stage protocol with a clear and repeatable process; at the same time, 3D cell spheres can complete self-assembly preparation within 24 hours.

[0045] 5. Completely solve the problem of skin source scarcity and achieve large-scale supply: This invention uses pluripotent stem cells as the source and obtains functional EKCs through three stages of directed induction differentiation; pluripotent stem cells have a near-unlimited self-renewal capacity and can be expanded on a large scale in vitro, providing an inexhaustible cell bank for EKCs preparation, completely getting rid of the skin source limitations of traditional autologous skin grafting and allogeneic skin grafting, meeting the large cell demand for wounds such as large-area thermal burns and diabetic foot ulcers, and is suitable for industrialized standardized production.

[0046] 6. Excellent biocompatibility and high safety: 3D cell spheres rely on the in situ ECM secreted by the cells themselves to construct the support network, eliminating the need for exogenous scaffold materials and avoiding the risks of heterogeneity and batch differences.

[0047] 7. Adaptable to various skin defect types and with a wide range of applications: 3D cell spheres can play an efficient repair role for acute and chronic skin defects such as large-area thermal burns, diabetic foot ulcers, and pressure sores. It not only solves the need for "rapid wound closure" in acute burns, but also addresses the problem of "long-term regeneration and repair" in chronic ulcers, providing a unified and efficient treatment path for different types of skin defects. Attached Figure Description

[0048] Figure 1 A shows the microscopic examination results of H9 hESCs after multiple generations of culture in Example 1. Scale bar: 200 μm. Figure 1 B represents the karyotype detection results of H9 hESCs after multiple generations of culture in Example 1; Figure 1 C represents the OCT4 and DAPI immunofluorescence staining results of H9 hESCs in Example 1. Scale bar: 200 μm. Figure 1 D represents the relative expression levels of PAX6 in ectoderm cells, brachyury, CD56, and CD184 in mesodermal cells, and SOX17 and FOXA2 in endodermal cells after the H9 hESCs of Example 1 underwent a trigerm layer differentiation experiment. Figure 2 A is a flowchart of the three-stage induction scheme in Example 1; Figure 2B shows the cell morphology of 0D (uninduced H9 hESCs), 5D, 10D, and successfully induced EKCs during the differentiation induction phase of Example 1. Scale bar: 200μm. Figure 2 C represents the expression of cell markers Δnp63, K18, K5, K14, Integrin β1, Integrin α6, LGR6, and K19 in 0D, 5D, 10D, and successfully induced EKCs during the differentiation process in Example 1. Figure 2 D represents the immunofluorescence staining results of K5, K14 and DAPI in EKCs of Example 1. Scale bar: 100 μm. Figure 2 E is a scanning electron microscope image of EKCs from Example 1; Figure 3 A shows a microscopic image of EKCs and a microscopic image of 3D cell spheres from Example 1. Scale bar: 100 μm. Figure 3 B represents the HE staining results of 3D cell spheres prepared in Example 4 when the EKCs:HFBs ratio was 1:5 or 5:1. Scale bar: 100 μm. Figure 3 C represents 5×10 in Example 3. 4 1×10 5 and 1.5×10 5 3D cell spheroid staining results for live and dead cells, scale bar: 100μm; Figure 3 D represents the K5, VIMENTIN, and DAPI immunofluorescence staining results of 3D cell spheres prepared in Example 4 when the EKCs:HFBs ratio was 3:1. Scale bar: 100 μm. Figure 3 E represents the COL I and DAPI immunofluorescence staining results of 3D cell spheres prepared in Example 4 when the EKCs:HFBs ratio was 3:1. Scale bar: 100 μm. Figure 4 A represents the percentage of K5 and K14 positive cells in p2 EKCs of Example 2; Figure 4 B represents the EKCs proliferation capacity detection results from Example 2; Figure 4 C represents the HE staining result of EKCs after in vitro induced differentiation 20 days after Example 2. Scale bar: 20 μm. Figure 4 D shows the K14 and K10 immunofluorescence staining results of the multilayered epidermal tissue in Example 2. Scale bar: 50 μm. Figure 4E represents the double immunofluorescence staining image of involucrin and DAPI, a nuclear marker, in the multilayered epidermal tissue formed by in vitro induced differentiation of EKCs in Example 2, and the double immunofluorescence staining image of loricrin and DAPI, a nuclear marker, in the multilayered epidermal tissue formed by in vitro induced differentiation of EKCs. Scale bar: 50 μm; Figure 4 F represents the transmission electron microscopy observation results of the multilayered epidermal tissue in Example 2; Figure 5 The results of observing the overall morphology and density of 3D cell spheres with different cell numbers in Example 3; Figure 6 A shows the morphological detection results of 3D cell spheres prepared by different proportions of EKCs and HFBs in Example 4. Scale bar: 100 μm. Figure 6 B shows the K5 and VIMENTIN immunofluorescence staining results of 3D cell spheres prepared in Example 4 when the EKCs:HFBs ratio was 10:1, 5:1, 1:1, 1:5, or 1:10. Scale bar: 50 μm. Figure 6 C represents the COL I and DAPI immunofluorescence staining results of 3D cell spheres prepared by different proportions of EKCs and HFBs in Example 4. Scale bar: 50 μm. Figure 7 A shows photographs of the wound healing status in Example 5 after different treatment groups, taken 5-20 days later. Figure 7 B shows the wound area change curves of the wound in Example 5 after 5-20 days of treatment in different groups; Figure 7 C represents the HE staining results of the wound tissue in Example 5 on days 10 and 20 after different treatment groups. Scale bars: 500 μm and 100 μm. Figure 8 A shows the immunofluorescence staining results of K14, K10 and DAPI in different groups 20 days after wound treatment in Example 5. Scale bar: 100 μm. Figure 8 B shows the immunofluorescence staining results of Involucrin, Loricrin and DAPI in the EKC+HFB Spheroids group 20 days after wound treatment in Example 5. Scale bar: 100 μm. Figure 8 C shows the K10 and DAPI immunofluorescence staining results of the 3D cell sphere treatment group (EKC+HFB Spheroids group) 10 days after wound treatment in Example 5. Scale bar: 100 μm. Figure 8 D represents the immunofluorescence staining results of K5, anti-human nuclear antibody, and DAPI in the 3D cell spheroids treatment group (EKC+HFB Spheroids group) on days 5 and 15 after wound treatment in Example 5. Scale bar: 200μm and 25μm. Figure 9 This is an in vivo imaging image taken 3 days after EKCs cell suspension was applied to a nude mouse wound for transplantation, as shown in Example 6. Figure 10 A is a light micrograph of the EKCs in vitro attached biomaterial in Example 6, scale bar: 100 μm; Figure 10 B is a cell liveness / death staining photograph after EKCs were attached to biomaterials in Example 6. Scale bar: 100 μm. Figure 10 C is a cytoskeleton staining photograph of EKCs after attaching biomaterials in Example 6. Scale bar: 100 μm. Figure 10 D is a live imaging photograph taken on the 3rd day after the transplantation of EKCs-attached biomaterials in Example 6; Figure 11 A is a gross photograph of the porcine acellular dermal matrix (ADM) material from Example 6; Figure 11 B is a HE staining photograph of dehydrated and freeze-dried porcine dermal matrix after Example 6, scale bar: 200 μm; Figure 11 C is a stained image of the EKCs skeleton loaded in ADM in Example 6. Scale bar: 50 μm. Figure 11 D is a live / dead staining photograph of EKCs cells loaded in ADM in Example 6. Scale bar: 100 μm. Figure 11 E is a live imaging photograph taken on day 3 after transplantation of EKCs loaded in ADM in Example 6; Figure 11 F is a HE-stained photograph of tissue from EKCs loaded in ADM in Example 6 on day 3 post-transplantation, scale bar: 100 μm; Figure 11 G represents the immunofluorescence staining of keratin and DAPI in tissues of EKCs loaded in ADM in Example 6 on day 3 post-transplantation. Scale bar: 100 μm. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0050] Example 1: Preparation of 3D cell spheres 1. In vitro induction of pluripotent stem cells to differentiate into keratinocytes (EKCs). In this embodiment, the pluripotent stem cells are selected as H9 human embryonic stem cells (H9 hESCs).

[0051] (1) Embryonic stem cell culture: H9 embryonic stem cells were seeded on matrix gel-coated cell culture plates and the mTeSR plus medium (stemcell) was changed daily. When the cells grew to a suitable density, they were passaged using relesr digestion solution (stemcell) and mTeSR plus medium containing 10 μM Y27632 (stemcell).

[0052] To ensure the smooth progress of the subsequent induction process, the state, karyotype, and pluripotency of H9 hESCs were first detected.

[0053] Cell condition was examined under a microscope, and the results were as follows: Figure 1 As shown in Figure A, the H9 hESCs of well-formed human embryonic stem cells exhibit clonal growth in clusters with tight intercellular connections, clear cluster edges, and no differentiated cells visible at the edges. Under high magnification, distinct cell nuclei are visible, exhibiting a large nucleocytoplasmic ratio.

[0054] Karyotype test results as follows Figure 1 As shown in Figure B, the number and morphology of H9 hESCs chromosomes after multiple generations of culture remain consistent with the standard human karyotype, indicating that the genome remains stable during long-term culture.

[0055] To detect the pluripotency of H9 hESCs, immunofluorescence staining was first performed on the intracellular pluripotency marker OCT4. The steps were as follows: Cells in the culture plate were fixed with 4% paraformaldehyde for 15 min, permeabilized with strong immunostaining permeabilization buffer (Beyotime) for 10 min, blocked with immunostaining blocking buffer (Beyotime) for 15 min, incubated overnight at 4°C after adding OCT4 primary antibody diluted to the official recommended concentration, incubated at 37°C for 50 min after adding diluted secondary antibody, stained with DAPI (SolarBio) for 10 min, and then mounted for observation. PBS was used to wash three times between all steps. The immunofluorescence staining results are shown below. Figure 1 As shown in Figure C, cells were found to highly express OCT4.

[0056] The steps for detecting the pluripotency of H9 hESCs are as follows: In vitro induction of embryonic stem cell differentiation into three germ layers was performed using the STEMdiff™ Trilineage Differentiation Kit (stemcell cat#05230). Healthy embryonic stem cells were seeded at different densities and treated with the appropriate reagents. Induction of mesoderm and endoderm was completed on day 5, and ectoderm induction on day 7. Next, relative gene expression levels were detected by RT-qPCR. The specific method is as follows: RNA was extracted from the mesoderm, endoderm, and ectoderm cells, as well as undifferentiated H9 hESCs, using an RNA extraction kit (AG21023). RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit (AG11706). The cDNA was then analyzed using the corresponding dye system (AG11701), with GAPDH as an internal control. A 2... -ΔΔCT The relative gene expression levels were calculated using the following methods; PAX6 was used as the detection index for ectoderm cells, brachyyury, CD56, and CD184 were used as detection indexes for mesoderm, and SOX17 and FOXA2 were used as detection indexes for endoderm. The detection results are as follows: Figure 1 As shown in Figure D, PAX6 is significantly expressed in the ectoderm, indicating that H9 hESCs can successfully differentiate into ectoderm precursor cells, providing direct evidence for subsequent induction of keratinocytes (ectoderm-originating cells). Brachyury, CD56, and CD184 are significantly expressed in the mesoderm, verifying that H9 hESCs can differentiate into mesodermal lineage cells. SOX17 and FOXA2 are significantly expressed in the endoderm, demonstrating that H9 hESCs possess endoderm differentiation potential. Therefore, H9 hESCs have the ability to differentiate into all three germ layers.

[0057] (2) EKCs-induced differentiation: such as Figure 2As shown in Figure A, a three-stage induction protocol was adopted. The first stage used DKSFM (ThermoFish) medium supplemented with 0.3 μg / mL RA (Sigma) and 25 ng / mL BMP4 (R&D Systems), and this stage was maintained for 5 days. The second stage used CNT 07 (Cellntec) medium supplemented with 20 ng / mL EGF (Sigma), and was also maintained for 5 days. The third stage used CNT 07 medium supplemented with 10 μM y27632, and this stage could be maintained for 5 days or longer. After the first stage, if the cell density was too high, normal passage was performed, and the cells were passaged to plates coated with 15 μg / mL collagen IV (Sigma). This process used type IV collagen (collagen IV) for selection, and only cells that successfully differentiated could adhere to the plate. The third stage medium was then used until the cells exhibited typical keratinocyte morphology (paving stone-like). The cell morphology of 0D (uninduced H9 hESCs), 5D, 10D, and successfully induced EKCs during the above-mentioned induction differentiation stages is as follows: Figure 2 As shown in B.

[0058] Gene expression was detected in successfully induced EKCs at 0D, 5D, and 10D during the above-mentioned induction differentiation process. The detection method was the same as that described in RT-qPCR. The genes detected included Δnp63, the earliest marker of the epidermal lineage; K18, a marker of monolayer epithelium; K5 and K14, markers of basal keratinocytes; and Integrin β1 and LGR6, markers of epidermal stem cells. The detection results are as follows: Figure 2 As shown in Figure C, Δnp63, one of the TPp63 subtypes, was found to be strongly expressed as the earliest marker of the epidermal lineage at day 5 of differentiation and remained highly expressed throughout the differentiation process. K18, as a marker of monolayer epithelium, was expressed in the early stages of differentiation and gradually decreased in the later stages. K5 and K14, as markers of basal keratinocytes, appeared in the mid-to-late stages of differentiation, indicating that embryonic stem cells had been successfully induced to differentiate into keratinocytes. At the same time, the epidermal stem cell markers Integrin α6, K19, Integrin β1, and LGR6 were all stably highly expressed in the mid-to-late stages of differentiation. Among them, Integrin α6 is a core marker molecule for the adhesion characteristics and stemness maintenance of epidermal stem cells, and K19 is a characteristic marker of epidermal stem progenitor cells. The expression trends of Integrin β1 and LGR6 are highly consistent with those of Integrin β1 and LGR6, jointly confirming that the differentiated keratinocytes have good stemness potential.

[0059] Furthermore, by detecting Vimentin and CD105 in the EKCs that were finally differentiated, it was demonstrated that the induced differentiated cells were almost free of fibroblasts, a common contaminating cell in the differentiation process.

[0060] Furthermore, the finally differentiated EKCs were subjected to immunofluorescence staining using the same method as above, but the primary antibody was replaced with anti-K5 and anti-K14 antibodies. The detection results are as follows: Figure 2 As shown in Figure D, the differentiated EKC cells were verified at the protein level to highly express K5 and K14.

[0061] In addition, the scanning electron microscope images of the finally differentiated EKCs are as follows: Figure 2 As shown in E, the presence of dense keratin intermediate filaments in the cell further confirms its identity as a keratinocyte.

[0062] The above experiments demonstrate that this embodiment successfully induced H9 hESCs to differentiate into EKCs.

[0063] 2. Preparation of 3D cell spheres like Figure 3 As shown in Figure A on the right, the EKCs and HFBs prepared above were mixed and cultured at a cell ratio of 3:1, with a total of 50,000 cells in the EKCs and HFBs, allowing them to spontaneously form 3D cell spheres. Figure 3 The left image shows that EKCs alone are difficult to form cell spheroids.

[0064] HFBs extraction protocol: Human skin tissue was rinsed three times with PBS containing working concentration of penicillin and fetal antibodies for 10 min each time. Fat and connective tissue were discarded, and the skin tissue was cut into 0.5 × 0.5 cm pieces. The pieces were placed in 2 U / mL (0.25%) dispase and incubated overnight (14–16 h) at 4°C. After peeling off the epidermis, the dermis was minced and digested with 0.25% trypsin at 37°C on a shaker at 180 rpm for 10 min. Digestion was terminated by adding a trypsin inhibitor. The mixture was filtered through an 80 μm filter. The filtrate was centrifuged at 1000 rpm for 5 min, resuspended in culture medium, and cultured in high-glucose DMEM medium containing 10% fetal bovine serum. Cells were passaged when confluence reached approximately 80%.

[0065] 3D cell spheroid culture protocol: After digesting EKCs and HFBs cells, dilute them separately to a concentration of 2.5 × 10⁻⁶ cells using their respective serum-free medium. 5 After mixing the cells at a ratio of 3:1, add 200 μL to each well of a low-adsorption U-shaped 96-well plate. Centrifuge the plate at 1000 rpm for 5 minutes at room temperature. After that, let the cells stand for 24 hours until they form spheroids. Once the cells have formed spheroids, they can be used, or they can be cultured in the plate for another 1-3 days.

[0066] Example 2: EKCs can be used to construct epidermis with a multilayered structure in vitro. To achieve the goal of rebuilding the epidermis, a large number of seed cells are needed, and most importantly, these cells must possess the functions of normal epidermal cells. Therefore, this example tested the induction efficiency and proliferation capacity of EKCs in Example 1.

[0067] First, the efficiency of induced differentiation was assessed using two classic keratinocyte markers, K5 and K14. The percentage of K5 and K14-positive cells in p2 EKCs was determined by flow cytometry. The results are shown below. Figure 4 As shown in Figure A, it was found that the average percentage of K5-positive cells in p2 EKCs reached over 93%, and the percentage of K14-positive cells reached over 90%.

[0068] Furthermore, the proliferation capacity of EKCs was detected using the computational method mentioned by Ruiz-Torres. The specific process is as follows: After cell differentiation and maturation, 4×10 5 Cells were seeded in six-well plates, harvested after 4 days, counted, and then 4 × 10⁶ cells were seeded. 5 Cells were seeded into new six-well plates until cell proliferation significantly decreased. Cell doubling time was then calculated using the formula PDT = 4 × log(2) / log(Total cell number / 400000), and the number of cell divisions per generation was calculated using the formula n = 4 / PDT. The results are as follows: Figure 4 As shown in Figure B, EKCs obtained by H9 hESCs-induced differentiation exhibited excellent proliferation capacity within 5 generations.

[0069] Furthermore, this embodiment verifies whether the EKCs induced in Example 1 possess the ability to form stratified epidermis under suitable conditions, similar to normal EKCs. This is crucial for the subsequent construction of functionalized epidermis in vivo. Keratinocytes were induced to differentiate in vitro for 20 days using a transwell chamber to simulate the in vivo gas-liquid interface environment. HE staining was performed after 20 days. The HE staining procedure was as follows: tissue samples were fixed with 4% paraformaldehyde, dehydrated, embedded, and prepared into paraffin blocks. After sectioning, HE staining was performed using a SolarBio HE staining kit according to the instructions. Afterward, the slides were mounted with degreased resin and the tissue morphology was observed under a microscope. The detection results are as follows: Figure 4 As shown in Figure C, it was found that after about 20 days of in vitro differentiation, EKCs could be observed to have a multilayered epidermal structure through HE staining.

[0070] Furthermore, the stratified epidermal tissue was stained with immunofluorescence, using the same method as in Example 1, but the primary antibodies were replaced with anti-K14 and anti-K10 antibodies. The detection results are as follows: Figure 4 As shown in Figure D, K14 was found to be highly expressed in basal cells, while K10 was expressed above the basal layer, a distribution consistent with that of the epidermis under physiological conditions. Figure 4 E-scans showed that Involucrin exhibited widespread and uniform positive expression throughout the entire stratified epidermis, indicating that the EKCs induced by this invention can stably initiate the epidermal terminal differentiation process. Loricrin showed specific high expression in the upper terminal differentiation region of the stratified epidermis, and its distribution pattern highly matched the differentiation pattern of normal human physiological epidermis, fully demonstrating that the EKCs obtained by the three-stage induction protocol of this invention possess complete epidermal differentiation potential, can complete the entire process from directed differentiation to terminal maturation, and can construct a stratified epidermal structure with complete barrier function, laying a functional foundation for high-quality epidermal regeneration through subsequent in vivo wound transplantation.

[0071] Desmosomes are one of the most important intercellular junction structures in the epidermis, ensuring the tight and stable arrangement of keratinocytes. Therefore, this embodiment uses transmission electron microscopy to detect the presence of desmosome structures in the aforementioned multilayered epidermal tissue. The specific detection steps are as follows: Epidermal tissue cultured for 4 weeks was pre-fixed for 2 hours with pre-cooled 2.5% glutaraldehyde; rinsed three times with 0.1 M phosphate buffer, 15 minutes each time; then post-fixed with 1% osmium tetroxide for 2 hours; the fixed sample was dehydrated with a gradient of 50%-100% ethanol for 15 minutes each time; next, the sample was transitioned with propylene oxide, then infiltrated in a mixture of epoxy resin and propylene oxide, and finally embedded in pure epoxy resin and polymerized at 60°C for 48 hours; ultrathin sections with a thickness of approximately 70 nm were prepared using an ultramicrotome; the sections were double-stained with uranyl acetate and lead citrate, and then observed and images were acquired under a transmission electron microscope. The detection results are as follows: Figure 4 As shown in Figure F, clear desmosome structures were observed between cells in the aforementioned stratified epidermal tissue, further demonstrating that the EKCs induced to differentiate in Example 1 possess the function of normal keratinocytes.

[0072] Example 3: Screening of 3D cell spheroid cell count Since the formation of 3D cell spheroids is mainly related to fibroblasts, when the spheroids become too large, the cells in the center may undergo apoptosis due to insufficient nutrient supply. Therefore, in this embodiment, the total number of EKCs and HFBs in Example 1 will be changed to 5 × 10⁻⁶ cells. 3 1×10 4 2×10 4 5×10 4 1×10 5 1.5×10 5 3D cell spheres were prepared according to the method in Example 1.

[0073] First, 3D cell spheres with varying cell numbers were observed using an inverted microscope to assess their overall morphology and density. The results are as follows: Figure 5As shown, the volume of the 3D cell spheroid gradually increases with the increase of cell number: when the cell number is 5 × 10⁻⁶. 3 and 1×10 4 At this stage, the spherical structure is incomplete or irregular, consisting of loose clusters of small cells with loose edges that are easily detached, and without a distinct dense core; when the cell number is 2 × 10⁻⁶ 4 At this stage, the spherical structure is initially complete, with relatively loose edges but no breakage; when the cell number is 5 × 10⁶ 4 At 1 × 10⁻⁶, the spherical structure is intact, the surface is smooth, and the internal cells are densely arranged without looseness or cracks; when the cell number is 1 × 10⁻⁶. 5 At 1.5 × 10⁻⁶, the spherical structure was intact but the surface was slightly rough, and some samples showed slight wrinkles at the edges; when the cell count was 1.5 × 10⁻⁶. 5 At that time, the spherical structure was taut, and some samples showed obvious cracks at the edges, and even local cell shedding.

[0074] Furthermore, based on the above microscopic examination results, 2×10⁻⁶ was initially selected. 4 5×10 4 1×10 5 and 1.5×10 5 The next step, live / dead cell staining detection, was performed on 3D cell spheres containing a certain number of cells. The specific procedure was as follows: The 3D cell spheres were washed twice with PBS, and a sufficient amount of prepared PI working solution (dead cell dye, red fluorescence) was added, ensuring the cell spheres were completely submerged. The cells were incubated at room temperature for 10 minutes. The PI working solution was removed, and the cells were gently washed once with sufficient PBS to remove the supernatant. A sufficient amount of prepared AM working solution (live cell dye, green fluorescence) was added, and the cells were incubated at room temperature for 20 minutes. After washing with PBS, the cells were observed using a laser confocal microscope. The observation results are as follows: Figure 3 As shown in Figure C, when the cell number is 2 × 10⁻⁶ 4 At a certain time, the 3D cell spheroids showed weak red fluorescence, no apoptotic aggregation in the center, and complete green fluorescence coverage; when the cell number was 5×10⁻⁶, the 3D cell spheroids exhibited weak red fluorescence, no apoptotic aggregation in the center, and complete green fluorescence coverage; 4 At this time, red fluorescence is sporadically distributed, with only a few scattered dead cells at the very center of the cell spheroid, while green fluorescence uniformly covers most of the area; when the cell number is 1×10⁻⁶, red fluorescence is scattered throughout, with only a few dead cells at the very center of the cell spheroid, while green fluorescence uniformly covers most of the area; when the cell number is 1×10⁻⁶... 5 At this time, red fluorescence significantly increased, a dense apoptotic core formed in the central region, and green fluorescence was only distributed on the surface; when the cell number was 1.5 × 10⁻⁶, red fluorescence increased significantly, and a dense apoptotic core formed in the central region, while green fluorescence was only distributed on the surface. 5 At this time, red fluorescence is diffused extensively, with large-scale apoptosis in the center, while green fluorescence is only present in a thin layer of surface cells. Therefore, it can be seen that 100,000 (1×10⁻⁶) cells... 5 ) and 150,000 (1.5 × 10 5 By day 3, a cell spheroid composed of 50,000 (5 × 10⁶) cells already contained a large number of dead cells, and the central cells had undergone apoptosis; 4The number of dead cells inside a cell sphere composed of 100 cells is relatively small, and the number of dead cells will decrease as the number of cells is further reduced. However, considering that the spheres are too small for subsequent operations, it is ultimately preferable to use 50,000 cells self-assembled in each cell sphere.

[0075] Example 4: Screening of cell ratios of EKCs and HFBs The mixing ratio of EKCs to HFBs is directly related to the density of the 3D cell spheres. In this embodiment, the mixing ratio of EKCs to HFBs in Example 1 will be changed. Specifically, EKCs:HFBs = 10:1, 5:1, 3:1, 1:1, 1:3, 1:5 or 1:10, and 3D cell spheres will be prepared according to the method of Example 1.

[0076] First, the morphology and structural density of the different 3D cell spheres were tested. The overall morphology and whether the cell spheres of each proportion group were easily broken were observed under an inverted microscope. Then, HE staining was performed. The HE staining procedure was the same as in Example 1. The density of cell arrangement and the gaps inside the cell spheres were observed.

[0077] Combining morphological and structural compactness test results (such as...) Figure 6 As shown in Figure A) and the HE staining results, it is evident that when EKCs:HFBs = 10:1 or 5:1 ( Figure 3 When EKCs:HFBs = 3:1 or 1:1, the spherical structure is loose, with incomplete edges, no obvious dense core, and is easily broken; when EKCs:HFBs = 1:3, the spherical structure is complete, dense, and not easily broken; when EKCs:HFBs = 1:5, the spherical structure is complete, dense, and has strong mechanical resistance; when EKCs:HFBs = 1:5, the spherical structure is not easily broken. Figure 3 (Left figure B) or at a ratio of 1:10, the spherical structure is dense, the volume is small, the surface is tight, and it forms a dense small mass, but the texture is hard.

[0078] Furthermore, the cell distribution of 3D cell spheroids at different ratios was detected by immunofluorescence staining. The detection procedure was as described in Example 1, and the primary antibodies were K5 antibody and VIMENTIN antibody. The detection results are as follows. Figure 3 D and Figure 6As shown in Figure B, when the ratio of EKCs to HFBs is 10:1 or 5:1, EKCs are dispersed without a continuous outer layer, and HFBs are few in number and scattered. When the ratio of EKCs to HFBs is 3:1 or 1:1, EKCs form a continuous and complete cell layer on the outside of the cell spheres; HFBs aggregate into clusters inside and are evenly distributed. When the ratio of EKCs to HFBs is 1:3, EKCs are scattered in small clusters on the surface and inside of the cell spheres; HFBs are dominant. When the ratio of EKCs to HFBs is 1:5 or 1:10, the cell spheres lack a complete outer layer structure, with HFBs being the main component and densely distributed, while EKCs are sporadically distributed on the surface or inside. Simultaneously, the distribution of extracellular matrix in 3D cell spheres with different ratios was detected by immunofluorescence staining. The detection procedure was the same as in Example 1, using type I collagen (COL I) antibody as the primary antibody. The detection results are shown in Figure 3E. Figure 6 As shown in Figure C, when the ratio of EKCs to HFBs is 10:1 or 5:1, the fluorescence signal of COL I is extremely weak, and no continuous extracellular matrix network is formed, failing to provide effective structural support for the cells. When the ratio of EKCs to HFBs is 3:1 or 1:1, the fluorescence signal intensity of COL I is moderate, forming a continuous and uniform network structure inside the cell spheroid, completely encapsulating the internal HFBs and the outer EKCs, providing stable mechanical support and a suitable growth microenvironment for the cell spheroid. When the ratio of EKCs to HFBs is 1:3, 1:5, or 1:10, the fluorescence signal of COL I is extremely strong, and the extracellular matrix excessively accumulates within the spheroid, forming an extremely dense network structure, which restricts the subsequent migration, proliferation, and differentiation functions of EKCs. Therefore, when the proportion of HFBs is too low, it is impossible to effectively form a firm spherical structure or secrete sufficient extracellular matrix to build a support network. The prepared cell spheres are loosely structured and easily break during transfer. When the proportion of HFBs is too high, EKCs cannot form a continuous and complete cell layer outside the spherical structure, and excessive extracellular matrix will limit the function of EKCs. Therefore, the preferred EKCs:HFBs ratio is 1 to 3:1. Considering that the ultimate goal is to transplant EKCs, it is necessary to maximize the number of EKCs to improve subsequent transplantation efficiency, while ensuring that the cell spheres have a stable structure and suitable extracellular matrix support. Therefore, the optimal EKCs:HFBs ratio is 3:1.

[0079] Based on the above experimental results, the optimal ratio of EKCs to HFBs is 1 to 3:1.

[0080] Example 5: 3D Cell Ball Therapy for Skin Injuries The animals used in this embodiment were 6-week-old NSG immunodeficient mice (purchased from Southern Modeling Company (Shanghai, China)). The experimental procedures were strictly carried out in accordance with relevant regulations and medical ethics requirements. After anesthesia with 0.3% sodium pentobarbital via intraperitoneal injection, the skin on the back was shaved off, and after disinfection with povidone-iodine, a full-thickness skin defect with a diameter of 0.8 cm was created next to the spine. The experimental animals were randomly divided into 5 groups: Blank Control (50 μL of serum-free mixed culture medium was added to the wound), EKCs-only Spheroids group (50 μL of cell spheres containing 50,000 EKCs was added to the wound), HFBs-only Spheroids group (50 μL of cell spheres containing 50,000 HFBs was added to the wound), and EKC-HFB mixed cell sphere treatment group (EKC+HFB Spheroids, self-assembled 3D cell spheres containing 50,000 cells and an EKC:HFBs ratio of 3:1 were uniformly transplanted into the wound (preparation method is the same as in Example 1)). Each group was secured with bacterial cellulose, hydrocolloid dressing, and elastic bandage sequentially from the inside out. The wound was observed and dressings changed every 3 days post-surgery. The bacterial cellulose dressing was kept moist throughout the experiment. Wound healing was photographed and wound area changes were recorded at 0, 5, 10, 15, and 20 days after cell transplantation. Simultaneously, samples were taken from anesthetized mice for HE staining, K14 immunofluorescence staining, and K5 and anti-human nuclear antibody double-labeled immunofluorescence staining, using the methods described in Example 1.

[0081] The general wound healing situation and wound area change curves are shown in Figures 7A-B. The results showed that the wound healing speed of the EKC-HFB mixed cell sphere treatment group was significantly faster than that of all other groups. The wound began to shrink significantly 5 days after surgery, the wound area shrank significantly 10 days after surgery, and 100% complete closure was achieved 20 days after surgery. The healing speed of the HFBs single cell sphere group was faster than that of the EKCs single cell sphere group. The healing speed of the EKCs single cell sphere group and the HFBs single cell sphere group was significantly slower than that of the EKC-HFB mixed cell sphere treatment group. Large areas of unhealed wounds still existed 10 days after surgery, and obvious unepithelialized areas still remained 20 days after surgery. The blank control group had the slowest healing process, and a large number of wounds were still not completely closed 20 days after surgery. Meanwhile, it was observed that mice in the EKC-HFB mixed cell sphere treatment group had formed a continuous epidermis before the wound was completely healed, achieving temporary wound closure. Although the granulation tissue at the bottom was still in the proliferation stage and the newly formed epidermis was still relatively fragile, this rapid wound barrier reconstruction significantly accelerated the overall healing process, while no such phenomenon was observed in the other groups.

[0082] Figure 7C shows the HE staining results of the wound tissue on postoperative days 10 and 20. On day 10, the EKC-HFB mixed cell sphere treatment group had formed a continuous and complete epidermal structure, except that the epidermal cells in the central area of ​​the wound had not yet reached the ordered level of normal physiological state. In contrast, the blank control group, the EKCs single-cell sphere group, and the HFBs single-cell sphere group failed to achieve wound closure, and no continuous epidermal structure was formed, with only a small amount of epithelialization in the wound edge area. On day 20, the skin structure of the EKC-HFB mixed cell sphere treatment group was intact, with no residual wound, and the epidermal cells were arranged in an orderly manner, possessing a multilayered structure consistent with normal skin, and the granulation tissue in the dermis was arranged regularly. In contrast, the other groups still had some areas that had not completed re-epithelialization, with discontinuous and uneven epidermal structure, disordered granulation tissue proliferation, and no complete multilayered epidermis.

[0083] Figure 8C shows the results of K14 immunofluorescence staining of the wound tissue on the 10th day after surgery. It can be clearly observed that a continuous K14 positive epidermal layer has formed in the center of the wound in the EKC-HFB mixed cell sphere treatment group. In contrast, no K14 positive cells appeared in the center of the wound in the blank control group, the EKCs single cell sphere group, and the HFBs single cell sphere group. Only a few scattered positive signals were present at the wound edge. Figure 8A shows the immunofluorescence staining results of K14 and K10 in the wound tissue on postoperative day 20. In the wound tissue of the EKC-HFB mixed cell sphere treatment group, K14 was continuously and uniformly linearly highly expressed in the basal layer of the regenerated epidermis, forming a complete basal layer structure, which provides a stable proliferative potential guarantee for the long-term homeostasis of the epidermis. K10 was specifically expressed in the spinous and granular regions above the basal layer, showing a clear layered distribution. Together with the K14 expression area, it formed a layered structure that completely conforms to the physiological laws of normal skin. This proves that the transplanted EKC-HFB mixed cell spheres not only achieved complete re-epithelialization of the wound, but also promoted the orderly differentiation and maturation of the regenerated epidermis, successfully constructing a structurally complete and functionally sound multilayered epidermal tissue. In stark contrast, no positive signals of K14 and K10 were detected in the blank control group and the EKCs monocellular spheroid group; the HFBs monocellular spheroid group only showed sporadic and scattered positive signals of K14 and K10 in the wound margin area, with no continuous positive expression in the central area of ​​the wound and no orderly epidermal layering structure. This proves that the other groups did not achieve complete reepithelialization of the wound and could not form a regenerated epidermis with normal physiological structure and barrier function, and their wound repair effect was far inferior to that of the EKC-HFB mixed cell spheroid treatment group of this invention. On the 20th day after surgery, immunofluorescence staining of Involucrin and Loricrin in the wound tissue was performed. The staining results of the EKC-HFB mixed cell spheroid treatment group are as follows. Figure 8As shown in Figure B, Involucrin exhibits continuous and uniform positive expression in the spinous and granular regions of the regenerated epidermis, indicating that the transplanted cells can stably initiate the epidermal terminal differentiation program and complete the mid-stage differentiation and development of the epidermis. Loricrin shows a specific, strong, layered positive distribution in the upper terminal differentiation region of the regenerated epidermis, and its expression pattern perfectly matches the distribution pattern of normal human physiological epidermis, proving that the regenerated epidermis has completed the entire cycle of terminal differentiation and maturation, forming a keratinized structure with complete barrier function. In contrast, the blank control group, the EKCs monocellular spheroid group, and the HFBs monocellular spheroid group only showed extremely weak and scattered positive signals of Involucrin and Loricrin in the wound edge area, with no specific layered expression in the central area of ​​the wound, and no complete terminally differentiated epidermal structure was formed.

[0084] On postoperative days 5 and 15, K5 and anti-human nuclear antibody were used for double-labeled immunofluorescence staining. The anti-human nuclear antibody specifically recognized transplanted human cells. The staining results of the EKC-HFB mixed cell sphere treatment group are shown in Figure 8D. The results showed that on postoperative day 5, the human EKCs and HFBs in the EKC-HFB mixed cell sphere treatment group maintained an intact spherical structure, gradually undergoing cell separation and directional migration, with good cell survival. On postoperative day 15, the human EKCs in the EKC-HFB mixed cell sphere treatment group had formed a continuous multilayered epidermal sheet structure on the wound surface, while the human HFBs migrated to the dermal region below the EKCs, highly consistent with the epidermal-dermal hierarchical structure of natural skin. The positive anti-human nuclear antibody staining results strongly demonstrate that the human cells in the transplanted EKC-HFB mixed cell spheres can successfully survive and colonize long-term in the wound, differentiating to form a fully functional multilayered epidermal structure.

[0085] The above experimental results show that the EKC-HFB hybrid cell spheres of the present invention significantly improve the retention and survival rate of cells on the wound surface, achieve rapid wound colonization and high-quality epidermal regeneration, and greatly accelerate the wound healing process, providing a highly efficient and safe new cell therapy solution for acute and chronic skin defects.

[0086] Example 6: Comparison of the effects of EKCs on skin defects treated with different transplantation methods Example 2 demonstrates that EKCs possess the ability to proliferate and construct multilayered epidermis. This example explores an effective in vivo transplantation protocol for epidermal cells. This example uses a full-thickness skin defect model on the back of BALB / cNude athymic nude mice to study the therapeutic effect of EKC transplantation. Since T cells are the main force mediating xenograft rejection, the immune system of mice lacking T cells cannot recognize and attack human EKCs, thus providing an immune-exempt microenvironment for transplanted human cells. This allows for long-term observation of the survival, proliferation, and functional reconstruction of human cells in vivo.

[0087] First, an attempt was made to directly drop an EKCs suspension onto the wound surface, and the EKCs were stained with Dir fluorescence to observe cell survival in the wound using in vivo imaging technology. The results showed that, 3 days post-transplantation, no fluorescence indicating EKCs was observed on the wound surface (e.g., ...). Figure 9 As shown in the figure, this is likely because the fluid used in cell suspension transplantation is quickly absorbed by the dressing or flows away from the wound edge, resulting in cell loss. At the same time, since the wound is a harsh inflammatory environment containing a large number of proteases and free radicals, the single-cell suspension is directly exposed to this environment, and the cell membrane is easily damaged. As a result, a small number of remaining cells may die due to the drastic change in the living environment.

[0088] After attempting to directly apply EKCs suspension to the wound surface, this embodiment further explored preparing a cell suspension by mixing EKCs and HFBs in a preferred ratio of 3:1. Cells were labeled using the same Dir fluorescent staining method as with the pure EKCs suspension. This EKCs+HFBs mixed suspension was then applied to a full-thickness skin defect on the back of BALB / cNude athymic nude mice. The same bandaging, fixation, and postoperative management procedures were followed. On the third postoperative day, in vivo imaging was used to observe cell survival and colonization at the wound surface. The results showed almost no obvious fluorescence signal on the wound surface, with only very weak residual fluorescence detected at the wound edge. This result was not significantly different from simply applying the EKCs cell suspension, indicating that the EKCs+HFBs mixed suspension also failed to achieve effective cell colonization and survival at the wound surface. The reason for this is that, although HFBs that can secrete extracellular matrix were added to the suspension, in the single-cell suspension state, HFBs could not secrete enough extracellular matrix in a short time to form an effective support and adhesion network, and could not provide physiological protection and adhesion basis for EKCs. The mixed cells would still be lost in large quantities with wound exudate and dressing absorption, and the single-cell EKCs and HFBs would be directly exposed to the inflammatory environment of the wound. The cell membranes were easily damaged by proteases and free radicals in the wound, and the remaining few cells would die due to the drastic change in the living environment. The two could not play a synergistic repair role on the wound.

[0089] Therefore, a further consideration is to pre-culture EKCs on a biomaterial (a medical-grade cell carrier membrane provided by the Burn Department of Shanghai Changhai Hospital) to form a sheet-like structure before transplantation, thereby reducing the risk of loss and increasing their resistance to external environmental factors (such as...). Figure 10 As shown in A). Cell viability staining results showed that although a small number of dead cells were present, most of the EKCs adhering to the biological material were alive (e.g., ...). Figure 10 (As shown in B), this indicates that the material has good biocompatibility and is suitable for EKC adhesion and survival. Staining the cytoskeleton (e.g., [insert staining method here]) further demonstrates this. Figure 10 As shown in Figure C, EKCs are densely distributed along the surface of the material, with cells interconnected to form a monolayer structure. Dir staining was then applied to the EKCs, and they were seeded onto the aforementioned biomaterial and allowed to grow to [the desired growth level]. Figure 10 The degree of cell confluence in C was assessed in a BALB / cNude athymic nude mouse wound transplantation experiment. Results showed that, on day 3 post-transplantation, in vivo imaging of small animals indicated that EKCs failed to successfully colonize the wound (e.g., ...). Figure 10 (As shown in D). Based on in vivo experimental results, EKCs adhered well to the biomaterial during in vitro culture, possibly because the abundant nutrients and cytokines in the culture medium promoted EKC adhesion and survival. Without these factors, transplanted cells may die because they cannot firmly anchor to the biomaterial. Furthermore, when sheet-like structures adhere to a wound surface, if the adhesion is not tight enough or the wound surface is uneven, tiny gaps may remain. These gaps can quickly fill with exudate or blood, forming effusion. This effusion not only isolates cells from nutrient exchange with the wound surface but also easily leads to infection, thus significantly reducing the likelihood of cell survival.

[0090] The two experiments above showed that directly applying cell suspension to the wound or constructing a membrane for transplantation did not promote wound healing. Therefore, further research is needed to find a transplantation method that allows for closer contact with the wound and is more conducive to the survival and adhesion of EKCs. Acellular dermal matrix (ADM) retains the extracellular matrix components (ECM) and three-dimensional ultrastructure of natural dermis, such as collagen and elastin. Furthermore, compared to synthetic materials or simple hydrogels, ADM has greater plasticity, can adapt to changes in wound morphology, and provides near-physiological support for epithelial cells. Currently, decellularization technology is relatively mature. Decellularizing porcine dermis can remove immune targets such as cell membranes and nuclei while preserving the above advantages, greatly reducing immune rejection. A relatively pure acellular dermal matrix (such as...) was obtained through decellularization of porcine skin. Figure 11As shown in AB), it can solidify into a gel at 37°C. EKCs were mixed with 1.5% ADM to form a gel. The biocompatibility of the material and the cellular state were first tested in vitro. F-actin staining of the cytoskeleton showed that EKCs loaded with ADM had a clear cytoskeleton structure and good cell spreading after culture, indicating that ADM has good cell compatibility and can support the adhesion and growth of EKCs (e.g., as shown in AB). Figure 11 (As shown in C). Live and dead cell staining results showed that most EKCs exhibited green fluorescence (live cells), with only a few cells showing red fluorescence (dead cells), indicating that the cells were in good condition in the culture system (e.g., Figure 11 (As shown in D). Furthermore, compared to previous results of live-dead staining of EKCs on biological materials, the proportion of dead cells was significantly reduced, indicating that this ADM is more suitable for EKC survival.

[0091] After confirming the good biocompatibility of the prepared ADM, Dir-labeled EKCs were used for in vivo transplantation into BALB / cNude athymic nude mice. Results showed that on day 3 post-transplantation, the ADM gel mixed with cells adhered to the wound surface and showed positive results in in vivo imaging. However, once the ADM gel on the wound surface was wiped away, no fluorescence was observed (e.g., ...). Figure 11 As shown in Figure E: the left image shows the wound without the bottom dressing removed; the middle image shows the wound after all dressings have been removed but not wiped; the right image shows the wound after wiping with saline. The decreasing fluorescence indicates that most cells were lost with the removal of the dressing on top of the ADM. Although a small number of cells remained attached to the ADM after dressing removal, they did not successfully colonize the wound. To confirm this conclusion, further pathological analysis of the wound tissue revealed that no epidermal structure formation was observed in the HE staining results of the wound tissue sections (e.g., Figure 11 As shown in F), and at the same time, pankeratin immunofluorescence staining failed to observe the presence of epidermal components on the wound surface (e.g., as shown in F). Figure 11 (As shown in G). Based on this consideration, it is concluded that EKCs, as epidermal cells, tend to grow on the matrix surface to form a monolayer rather than penetrating deep into the matrix. If they were uniformly mixed into the matrix, most cells might be embedded deep within the matrix and die due to hypoxia; or they might be unable to migrate to the surface in time to form an epidermal layer. Furthermore, the large amount of chemical reagents used in the ADM treatment process may not have been completely removed in later processing, thus affecting the cells within. Figure 11As seen in Figure D, some dead cells emitting red fluorescence remained. To confirm whether the influence of residual chemical reagents was the cause, commercially available matrix gel for cell culture was used for transplantation in the same manner. Matrix gel is liquid at low temperatures, allowing for cell mixing; it solidifies into a gel at body temperature, perfectly encapsulating cells, providing extracellular matrix support, and preventing cell loss. Furthermore, matrix gel contains laminin, collagen IV, and growth factors, which can provide signals promoting epithelial cell survival and adhesion. Compared to self-prepared ADM, matrix gel has higher biocompatibility and better stability; however, the transplantation still did not yield ideal results, thus confirming the view that the transplantation method of mixing EKCs with exogenous extracellular matrix affects cell migration.

[0092] The results of the 3D cell sphere transplantation treatment experiment in Example 5, compared with the three transplantation methods in this example, show that the 3D cell sphere transplantation strategy fundamentally avoids the core problems of the three methods of dripping EKCs cell suspension, constructing EKCs cell sheets, and loading EKCs with exogenous extracellular matrix materials, and demonstrates better skin defect repair effect and clinical application potential.

[0093] The direct application of EKCs cell suspension presents challenges due to the ease with which cells are absorbed by the wound dressing and lost from the wound edge. Furthermore, the direct exposure of single cells to the harsh inflammatory environment of the wound makes their cell membranes susceptible to damage from proteases and free radicals, leading to rapid cell death. In this example, three days after transplantation, there was no fluorescent signal from EKCs on the wound surface, making cell colonization impossible. In contrast, 3D cell spheres possess a layered structure that mimics natural skin, with a continuous EKCs layer on the outside and aggregated HFBs and self-secreted ECM on the inside, forming a dense and stable spherical structure. The size and structural characteristics make them less susceptible to being washed away by wound exudate and blood flow after transplantation, significantly improving the retention and residence efficiency of cells at the site of injury. In Example 5, the transplanted human EKCs and HFBs successfully survived in vivo, maintaining a spherical structure at 5 days and gradually differentiating into a multilayered sheet structure at 15 days, effectively achieving cell colonization.

[0094] Although the EKCs+HFBs mixed suspension was added directly with the same cell ratio as in Example 5, the lack of stable structural support meant that the single-celled HFBs could not quickly secrete sufficient ECM to form a protective and supportive network in the harsh environment of the wound. The EKCs and HFBs could not form tight intercellular connections, nor could they resist changes in osmotic pressure and inflammatory impacts in the wound. Ultimately, they still could not escape the fate of massive loss and rapid death. Three days after the operation, there was almost no fluorescence signal in the wound, and cell colonization and epidermal regeneration could not be achieved. In Example 5, the 3D cell spheres were formed by the self-assembly of EKCs and HFBs in a 3:1 ratio. Internally, HFBs were pre-secreted to form a continuous ECM support network, while externally, a complete EKC layer was formed. This biomimetic structure prevents the cell spheres from being washed away by wound exudate after transplantation, allowing them to firmly adhere to the wound surface. Five days after transplantation, the human cells maintained their spherical structure and survived; after 10 days, continuous epidermal tissue was formed; and after 20 days, 100% wound closure and orderly epidermal arrangement were achieved. This comparison clearly demonstrates that simply mixing EKCs and HFBs does not guarantee their synergistic repair effect. The self-assembled spherical structure of the 3D cell spheres is the core factor ensuring HFBs secrete ECM, providing a physiological microenvironment for EKCs, and simultaneously achieving cell retention, survival, and functional performance. This is also the key to its high-efficiency skin defect repair compared to suspension transplantation.

[0095] While cell patch transplantation can achieve good adhesion between EKCs and biomaterials and form a monolayer structure in vitro, the cells cannot firmly anchor to the material and are prone to death after losing the support of nutrients and cytokines in the culture medium in vivo. Furthermore, the tiny gaps formed by the poor adhesion between the patch and the wound can be filled with tissue fluid and blood, forming effusion. This not only isolates the cells from the nutrient exchange with the wound but also easily leads to infection, ultimately resulting in the failure of cells to colonize successfully 3 days after transplantation. In contrast, 3D cell spheres are formed by cell self-assembly. The internal structure, composed of tight junctions, gap junctions, and cell-matrix interactions, forms a tissue-like three-dimensional microstructure, which can significantly reduce the stress response and apoptosis rate of cells during transplantation. At the same time, the in situ ECM secreted by the cells themselves provides immediate physiological support and protection for EKCs, effectively resisting changes in wound osmotic pressure and inflammatory shocks, avoiding cell death, and improving transplant survival rate. In Example 5, this structure ensured the function of EKCs in vivo, and a continuous epidermis was formed 10 days after the operation, which is far superior to the non-colonization effect of patch transplantation.

[0096] Regarding the method of loading cells onto exogenous ECM materials (porcine decellularized dermal matrix, matrix gel), EKCs, as epidermal cells, tend to grow on the surface of the matrix to form a monolayer. Cells uniformly mixed into the matrix often die due to hypoxia or cannot migrate to the surface in time to form an epidermal layer. Furthermore, homemade ADM has the problem of chemical reagent residues, which can further lead to cell death. Even commercially available matrix gels with higher biosafety levels are prone to cell loss when the material is removed, ultimately failing to form an epidermal structure on the wound. In contrast, 3D cell spheres rely on ECM secreted by the cells themselves to construct a support network. There is no risk of heterogeneity or chemical residues associated with exogenous materials, nor is there any hypoxia caused by cell embedding. The ECM components such as COL I secreted by HFBs inside form a continuous support network, providing a suitable microenvironment for the proliferation and differentiation of EKCs. It can also promote high-quality integration of the graft and the host by simulating epithelial-mesenchymal interactions. As seen in Example 5, the transplanted HFBs gradually migrate to the dermal layer beneath the EKCs, while the EKCs form a fully functional multilayered epidermal structure on the wound surface. 100% wound closure is achieved 20 days postoperatively, and the epidermal cells are arranged in an orderly manner. In contrast, the method of loading cells with exogenous ECM materials did not result in the formation of an epidermal structure or the presence of fluorescent signals of epidermal components in the wound tissue sections.

[0097] In summary, the 3D cell sphere transplantation approach, through the dual advantages of structure and microenvironment, solves the core pain points of the other three transplantation methods in terms of cell loss, survival, colonization, and epidermal regeneration. It comprehensively surpasses the above three transplantation methods in terms of cell survival efficiency, wound colonization ability, epidermal continuity and regeneration thickness, and overall biocompatibility. It is a safer, more efficient, and clinically valuable cell transplantation strategy for skin defect repair.

[0098] Example 7: Screening of Formulas for Three-Stage Differentiation Induction This embodiment aims to screen a culture medium system and inducing factor formulation that can efficiently and stably induce pluripotent stem cells to differentiate into functional keratinocytes (EKCs), and to determine the optimal parameters for the three-stage induction protocol, providing a stable and controllable seed cell induction system for the large-scale preparation of 3D cell spheres. In this embodiment, all parallel experimental groups maintained the same culture conditions as in Example 1, except for the culture medium and inducing factor variables to be screened. The optimal induction formulation was ultimately determined through cell morphology observation, detection of epidermal lineage-specific marker expression, and assessment of cell proliferation capacity and functional integrity.

[0099] 1. Screening of basal culture medium system for three-stage induction of differentiation In the preliminary experiments, our team attempted a stepwise induction protocol: “E8 medium (stemcell) + 5 ng / mL BMP4 + 1 μM RA for 7 days, DKSFM medium (Thermo Fisher) + EGF and FGF for 7 days, and pure DKSFM medium for 7 days.” This protocol failed to induce EKCs that met the functional requirements. Due to the presence of multiple variables in the experimental process, it was impossible to determine whether the induction failure was solely due to the conditions of the induction medium. Therefore, we redesigned a systematic three-stage medium screening experiment and conducted parallel verification of the basic mediums for each of the three stages of induction and differentiation.

[0100] The first stage is the epidermal lineage directed differentiation stage (induction days 0-5), with the core objective of efficiently inducing pluripotent stem cells into epidermal lineage precursor cells. Multiple parallel experiments have verified that this stage requires the simultaneous addition of both BMP4 and RA inducing factors to achieve directed differentiation of pluripotent stem cells into the epidermal lineage; the absence of either factor prevents the completion of the directed induction process. Regarding the basal culture medium for this stage, this example compares the induction effects of E8 medium and DKSFM medium in parallel. The results show that when DKSFM is used as the basal medium, cell morphology uniformity is better, the upregulation of the early epidermal lineage marker Δnp63 is more significant, and the directed differentiation efficiency is much higher than that of E8 medium. Therefore, DKSFM is determined to be the optimal basal culture medium for the first stage.

[0101] The second stage is the proliferation and expansion stage of immature keratinocytes (induction days 5-10). The core objective is to induce epidermal lineage precursor cells into immature keratinocytes while achieving large-scale cell proliferation and expansion. Initially, the team used only DKSFM as the basal medium for this stage, and hypothesized that CNT07 medium could meet the cell culture requirements of this stage. Therefore, this embodiment compared the induction effects of DKSFM and CNT07 media in parallel. The results showed that when CNT07 was used as the basal medium for this stage, cell proliferation was superior, the expression of keratinocyte-specific markers K5 and K14 was initiated earlier and at higher levels, and the cells could stably maintain a typical cobblestone morphology without fibroblast-like contamination. The overall induction effect was better than that of DKSFM medium. Therefore, CNT07 was determined to be the optimal basal medium for the second stage.

[0102] The third stage is the maturation and stemness maintenance stage of functional keratinocytes (days 10-15 of induction and subsequent passage culture). The core objective is to obtain functional EKCs with complete function and stable stemness, while ensuring the proliferative activity of cells during passage. This example compares the culture effects of three media: DKSFM, CNT07, and CNT NX E (an upgraded version of CNT07). The results show that all three media can support the in vitro culture of EKCs in this stage. Among them, CNT07 medium can better maintain the proliferative activity and differentiation potential of EKCs. The induced cells can still maintain excellent proliferative capacity within 5 passages, and the cell functional integrity and population uniformity are optimal. Considering the induction effect, operational stability and application cost, CNT07 is determined to be the optimal basal medium for the third stage.

[0103] In summary, after systematic screening and verification, the optimal combination of basic culture media for the three stages of pluripotent stem cell directed differentiation into EKCs is as follows: DKSFM medium for the first stage, CNT07 medium for the second stage, and CNT07 medium for the third stage.

[0104] 2. Screening and optimization of key inducing factors for three-stage differentiation induction Based on the above-mentioned optimal basal culture medium combination, this embodiment conducts univariate parallel validation on the types and working concentrations of key inducing factors in the three-stage induction system. Through epidermal lineage marker expression detection, cell proliferation capacity assessment, and cell function verification, the optimal inducing factor formulation for each stage is determined.

[0105] In the first stage of inducing factor optimization, the core functional components were BMP4 and RA. This example conducted parallel validation with three concentration gradients of BMP4: 5 ng / mL, 20 ng / mL, and 25 ng / mL. The results showed no significant difference in the directed differentiation effect of the epidermal lineage at the three concentrations, and all three effectively initiated the directed differentiation of pluripotent stem cells into the epidermal lineage. RA is the key core factor for directed differentiation in this stage; without RA, directed induction of the epidermal lineage cannot be completed. Experimental verification showed that a RA concentration of 0.3 μg / mL achieved the optimal directed differentiation effect, and its synergistic effect with BMP4 efficiently induced pluripotent stem cells to differentiate into epidermal lineage precursor cells. The final optimal inducing factor formulation for the first stage was determined to be: 0.3 μg / mL RA and 25 ng / mL BMP4 added to DKSFM medium.

[0106] In the second stage of inducing factor optimization, the core objective was to promote the proliferation and expansion of immature keratinocytes. This embodiment conducted univariate validation on two cytokines, EGF and FGF, with a final EGF concentration of 20 ng / mL and a final FGF concentration of 10 ng / mL. The induction effects of two parallel treatments were validated: "adding only EGF" and "adding both EGF and FGF." The results showed that both treatments supported cell proliferation and differentiation, with no significant difference in induction effects. Adding FGF did not significantly improve the induction effect. From the perspective of simplifying the treatment and controlling costs, the second stage did not require the addition of FGF; adding only EGF was sufficient to achieve the optimal proliferation and expansion effect. The optimal inducing factor formulation for the second stage was ultimately determined to be: 20 ng / mL EGF added to CNT07 medium.

[0107] In the third stage of inducing factor optimization, the core objective was to inhibit apoptosis and maintain cell stemness and adhesion. This example conducted parallel validation of the effect of adding the ROCK inhibitor Y27632, comparing the culture effects of two groups: "adding 10 μM Y27632" and "not adding Y27632". The results showed that adding 10 μM Y27632 significantly reduced the apoptosis rate, greatly increased the adhesion rate, and improved cell morphological uniformity. It effectively maintained the stemness and differentiation potential of keratinocytes, and maintained excellent proliferative activity during passage. The cell state was significantly better than the group without Y27632. Finally, the optimal inducing factor formulation for the third stage was determined to be: CNT07 medium supplemented with 10 μM Y27632.

[0108] In summary, this embodiment, through systematic screening and verification of culture media and inducing factors, ultimately determined the optimal three-stage induction system for the directed differentiation of pluripotent stem cells into EKCs as follows: Stage 1 (0-5 days): DKSFM medium supplemented with 0.3 μg / mL RA and 25 ng / mL BMP4; Stage 2 (5-10 days): CNT07 medium supplemented with 20 ng / mLEGF; Stage 3 (after 10 days): CNT07 medium supplemented with 10 μM Y27632. This induction system can efficiently and stably induce the directed differentiation of pluripotent stem cells into high-purity, highly active, and fully functional EKCs, providing a stable source of seed cells for the large-scale, standardized preparation of 3D cell spheres.

[0109] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A 3D cell sphere, characterized in that, It is formed by the self-assembly of keratinocytes and cells that can secrete extracellular matrix; the outer layer of the 3D cell sphere is a keratinocyte layer, and the inner layer is cells that can secrete extracellular matrix.

2. The 3D cell sphere as described in claim 1, characterized in that, The total number of keratinocytes and cells that secrete extracellular matrix is ​​20,000 to 100,000.

3. The 3D cell sphere as described in claim 2, characterized in that, The ratio of keratinocytes to cells that secrete extracellular matrix is ​​1 to 3:

1.

4. The 3D cell sphere as described in claim 1, characterized in that, The cells that can secrete extracellular matrix include any one of human fibroblasts, various types of mesenchymal stem cells, various types of endothelial cells, and chondrocytes.

5. The 3D cell sphere as described in claim 4, characterized in that, The cells that secrete extracellular matrix are human fibroblasts.

6. The method for preparing 3D cell spheres according to any one of claims 1 to 5, characterized in that, The keratinocytes are mixed with cells that secrete extracellular matrix to spontaneously form 3D cell spheres.

7. The preparation method according to claim 6, characterized in that, The keratinocytes are induced to differentiate from pluripotent stem cells.

8. The preparation method according to claim 7, characterized in that, The pluripotent stem cells include human embryonic stem cells and induced pluripotent stem cells.

9. The preparation method according to claim 7, characterized in that, The directed induction process employs a three-stage induction process: the first stage induces pluripotent stem cells into epidermal lineage precursor cells; the second stage induces epidermal lineage precursor cells into immature keratinocytes; and the third stage induces immature keratinocytes into functional keratinocytes.

10. Use of the 3D cell spheres as described in any one of claims 1 to 5 in the preparation of formulations that enhance the therapeutic effect on skin defects.

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