Differentiation medium for three-dimensional human epidermal organoids, preparation method and application thereof

Abstract

The application discloses a three-dimensional human epidermis organoid differentiation culture medium, a preparation method and application thereof, and belongs to the technical field of cell culture. The application provides a culture medium with clear chemical components and optimized proportioning, and combines a standardized culture method, so that human adult epidermis stem cells are stably and efficiently constructed into a three-dimensional human epidermis organoid with a complete epidermis layering structure (including a basal layer, a spinous layer and a keratin layer, and a granular layer), and meanwhile, key problems in the prior art, such as poor stability, high heterogeneity and incomplete epidermis layering differentiation, are solved. The culture system is compatible with two mainstream differentiation culture modes of air-liquid interface contact culture and suspension culture, and can realize stable epidermis layer differentiation, so that a more stable, repeatable and higher-physiological-relevance human epidermis organoid construction system is established.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically to a differentiation culture medium, preparation method and application of three-dimensional human epidermal organoids. Background Technology

[0002] As the largest organ in the human body, the skin, particularly its outermost epidermal tissue, possesses a complete layered structure and functional barrier consisting of the basal layer, spinous layer, granular layer, and stratum corneum. It plays an irreplaceable role in the body's protection, immune regulation, and hydration. In recent years, with the rapid development of organoid technology, the in vitro reconstruction of human skin tissue that highly simulates real-world conditions has become a cutting-edge research area in fields such as skin biology, wound repair and treatment, drug screening, and cosmetic safety evaluation.

[0003] Traditional two-dimensional cell culture models, while simple to operate, only allow human epidermal keratinocytes cultured in them to form single or multilayered epithelium. They cannot spontaneously establish a complete spatial structure from the basal layer to the stratum corneum, let alone form an effective functional barrier. These inherent structural and functional defects make it difficult to realistically simulate the complex processes of natural skin under physiological homeostasis or pathological conditions, greatly limiting their application value.

[0004] To overcome the limitations of two-dimensional culture, three-dimensional epidermal models have emerged. Currently, the mainstream techniques in this field mostly rely on Matrigel derived from mouse sarcoma as a three-dimensional scaffold for cell growth, supplemented by culture media containing serum or whose composition is not fully defined, to induce epidermal stratification through suspension or gas-liquid interface culture. However, this technical approach has several fundamental drawbacks that are difficult to overcome: for example, the lack of unified and precise standards for the types, concentrations, and timing of growth factors added in conventional culture media makes it easy for even cells from the same source to produce significant heterogeneity in different experiments: some epidermal organoids obtained can only form a basal layer and a thin stratum corneum, with the crucial spinous and granular layers incompletely developed or missing; some may exhibit excessive keratinization, leading to abnormal barrier function. In addition, in long-term culture exceeding four weeks, the model often experiences central necrosis due to insufficient nutrient and oxygen delivery, and the subsequent collapse of the stratified structure, ultimately making it difficult to stably and reproducibly obtain functional epidermal organoids with a complete four-layer structure and normal expression of key barrier proteins (such as Filaggrin and Loricrin).

[0005] In the prior art, there are known skin organoid differentiation media formulated based on Ham's F-12, such as the protocol described in the paper "Three-Dimensional Tissue Models of Normal and Diseased Skin, Mark W. Carlson, Addy Alt-Holland, Christophe Egles, Jonathan A. Garlick," published in the journal "Current Protocols in Cell Biology," Volume 41, Chapter 19.9, pp. 19.9.1–19.9.17, December 2008. However, this protocol contains 5% serum components, leading to uncontrollable differentiation processes and making it difficult to achieve stable and uniform large-scale production. Furthermore, the specific components and concentration ratios of commercially available CnT-PR-3D media are usually not fully disclosed, making it difficult for users to optimize and adjust according to specific experimental needs, and the price is relatively expensive, resulting in high costs for long-term or large-scale culture.

[0006] In summary, there is an urgent need in this field for a three-dimensional epidermal organoid culture medium with a clearly defined chemical composition, free of any animal-derived components, and capable of precisely inducing basal cells to differentiate sequentially into the spinous layer, granular layer, and stratum corneum in an orderly and terminal manner, much like program control. This is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a differentiation culture medium, preparation method, and application of three-dimensional human epidermal organoids. This culture system can stably induce human adult epidermal stem cells to differentiate into the spinous layer, granular layer, and stratum corneum (terminally forming keratinized squamous epithelium) within 20 days, constructing a complete skin barrier. The culture medium provided by the present invention has a well-defined composition and strong controllability, making it suitable for standardized production and large-scale in vitro construction of organoids. It has significant scientific research and clinical application value for advancing stem cell therapy for skin injuries, in vitro screening and evaluation of cosmetics and toxic drugs.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The primary objective of this application is to provide a differentiation culture medium for three-dimensional human epidermal organoids, using Ham's F-12 medium as the basal medium, and supplementing the basal medium with the following components at final concentrations: 2.4-9.6 mM L-glutamine, 24-96 μM adenine, 0.6-3 μM hydrocortisone, 12-48 ng / mL epidermal growth factor, 2-5 μM Y-27632, 5-10 μM A83-01, 12-48 nM triiodothyronine, 6-24 μg / mL insulin, 6-24 μg / mL transferrin, 1.2-4.8 nM progesterone, 1.08-2.16 mM calcium chloride, 0.3-1.2 mg / mL linoleic acid, and 0.3-1.2 mg / mL linoleic acid. mg / mL linolenic acid, 5.34-21.36 μg / mL alanine, 7.98-31.92 μg / mL asparagine, 8.82-35.28 μg / mL glutamate, 4.5-18.0 μg / mL glycine, 6.9-27.6 μg / mL proline, 6.3-25.2 μg / mL serine, 12.6-50.4 μg / mL arginine, 0.6-2.4 mM acetyl-L-cysteine, and 30-120 μM β-mercaptoethanol.

[0009] As a preferred technical solution, the differentiation medium uses Ham's F-12 medium as the basal medium, and the following components are added to the basal medium at final concentrations: 4 mM L-glutamine, 40 μM adenine, 1 μM hydrocortisone, 20 ng / mL epidermal growth factor, 5 μM Y-27632, 5 μM A83-01, 20 nM triiodothyronine, 10 μg / mL insulin, 10 μg / mL transferrin, 2 nM progesterone, 1.8 mM calcium chloride, 0.5 mg / mL linoleic acid, 0.5 mg / mL linolenic acid, 8.9 μg / mL alanine, 13.3 μg / mL asparagine, 14.7 μg / mL glutamate, 7.5 μg / mL glycine, 11.5 μg / mL proline, 10.5 μg / mL serine, 21 μg / mL arginine, 1 mM N-acetyl-L-cysteine, and 50 μM β-Mercaptoethanol.

[0010] Another object of this application is to provide a method for preparing the above-mentioned differentiation culture medium, comprising the following steps: (1) Dissolve L-glutamine, adenine, epidermal growth factor, insulin, transferrin, calcium chloride, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine ​​or β-mercaptoethanol in Ham's F-12 to prepare the corresponding stock solutions. (2) Dissolve hydrocortisone, A83-01, Y-27632, triiodothyronine, progesterone, linoleic acid or linolenic acid in DMSO to prepare corresponding stock solutions; (3) Add each mother liquor from steps (1) to (2) to Ham's F-12 according to the final concentration of each component in the culture medium to prepare differentiation culture medium.

[0011] Another object of this application is to provide the application of the above-described culture medium or the differentiation culture medium prepared by the above method, wherein the application is in any of the following directions: (1) Application in three-dimensional human epidermal organoid differentiation culture; (2) Application in the preparation of a three-dimensional human epidermal organoid differentiation culture kit; (3) Application in the preparation of three-dimensional human epidermal organoid in vitro models; (4) Application in stem cell therapy research for skin lesions or in vitro screening and evaluation of cosmetics and toxic drugs.

[0012] Another object of this application is to provide a kit for the differentiation and culture of three-dimensional human epidermal organoids, comprising the differentiation culture medium.

[0013] Another object of this application is to provide a method for culturing three-dimensional human epidermal organoids, comprising the following steps: using the aforementioned differentiation culture medium as a basis, inducing differentiation and culturing human adult epidermal stem cells by gas-liquid interface contact culture or suspension culture to prepare three-dimensional human epidermal organoids.

[0014] As a preferred technical solution, the specific process of the gas-liquid interface contact culture method is as follows: (a) Construction of a cell-free basal layer: In a Transwell chamber, type I rat tail collagen was neutralized to neutral using 0.1M sodium hydroxide solution for three-dimensional gelation to form a cell-free basal layer; (b) Establishment and pretreatment of the dermis: Human dermal fibroblasts were mixed with type I rat tail collagen and then subjected to a concentration of 1.5-2.5 × 10⁻⁶. 4 The cells / cm² were inoculated onto the cell-free basal layer. After culturing in culture medium for 3-5 days, the culture medium was removed and the mixture was allowed to stand for 15 minutes under sterile conditions. (c) Inoculation with human adult epidermal stem cells: Human adult epidermal stem cells were inoculated at a rate of 5-8 × 10⁻⁶. 4 The cells / cm² were seeded on the surface of the dermal layer obtained in step (b), left to stand for 15 min, and then placed in an incubator for 30 min. After that, they were cultured in amplification medium for 3-5 days. (d) Gas-liquid interface culture to induce differentiation: Add the differentiation medium according to any one of claims 1-2 to the lower chamber of the Transwell, while no medium is added to the upper chamber to establish a gas-liquid interface culture environment. Change the medium every day and continue culturing to induce the epidermis to form a complete layered structure. The specific process of the suspension culture method is as follows: (i) Divide 1-5×10 4 Individual adult epidermal stem cells were seeded in low-absorption multi-well plates and cultured using expansion medium to form cell clusters. (ii) Transfer the cell clusters formed in step (i) to the amplification medium for shaker suspension amplification culture; (iii) After the culture in step (ii) is completed, the culture medium is replaced with the differentiation culture medium described in any one of claims 1-2, and the suspension culture is continued to induce the differentiation and maturation of epidermal organoids.

[0015] Another object of this application is to provide: a three-dimensional human epidermal organoid cultured by the method.

[0016] Another object of this application is to provide: the application of the aforementioned three-dimensional human epidermal organoid, wherein the application is in any of the following directions: A: Application in the screening and development of skin-related drugs; B: Application in skin irritation, corrosivity and / or phototoxicity testing of cosmetics, pharmaceuticals and chemicals; C: Applications in the development of full-thickness skin organoids containing appendages such as hair follicles, sweat glands, and sebaceous glands; D: Applications in the development, screening, and pharmacological efficacy evaluation of drugs for skin diseases; E: Application in research on skin aging mechanisms and evaluation of cosmetic effects.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) In the culture medium of this invention, Y-27632 effectively prevents apoptosis and promotes the formation of three-dimensional structures by inhibiting the Rho / ROCK pathway, while A83-01 relieves the inhibitory effect on epithelial cell differentiation by inhibiting the TGF-β signaling pathway. The two work synergistically to significantly improve the formation efficiency and differentiation potential of organoids. On this basis, the epidermal differentiation process is finely regulated by triiodothyronine and progesterone, the long-term culture stability is maintained by a complex antioxidant system composed of N-acetyl-L-cysteine ​​and β-mercaptoethanol, and comprehensive nutritional support is provided by supplementing various specific amino acids. The synergistic effect of each component solves the technical bottlenecks in the survival, formation and differentiation of epidermal organoids. It can stably induce human adult epidermal stem cells to differentiate into spinous layer, granular layer and stratum corneum (terminally forming keratinized squamous epithelium) within 20 days, construct a complete skin barrier, and successfully obtain structurally complete and functionally mature epidermal organoids.

[0018] (2) The culture medium provided by the present invention has a clear composition and strong controllability, and is suitable for standardized production and large-scale in vitro construction of organoids. It has important scientific research and clinical application value for promoting stem cell therapy for skin damage, in vitro screening and evaluation of cosmetics and toxic drugs. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 Typical morphological structure of three-dimensional human epidermal organoids prepared by the suspension culture method in Example 1.

[0021] Figure 2 The morphological characteristics of three-dimensional human epidermal organoids prepared by the suspension culture method in Example 2 are as follows:

[0022] Figure 3 Example 2: Expression of layered biomarkers in three-dimensional human epidermal organoids prepared by suspension culture and normal human skin tissue samples.

[0023] Figure 4 For: Expression and localization of stratum corneum-specific markers in three-dimensional human epidermal organoids prepared in Example 3 and normal human skin tissue samples.

[0024] Figure 5 Example 4: Expression of epidermal layering-related markers in three-dimensional human epidermal organoids and normal human skin tissue samples prepared by gas-liquid interface contact culture.

[0025] Figure 6 Example 5: Expression of epidermal basal layer markers and terminal differentiation markers in three-dimensional human epidermal organoids and normal human skin tissue samples prepared by gas-liquid interface contact culture.

[0026] Figure 7 The results of hematoxylin-eosin (HE) staining of three-dimensional human epidermal organoids and normal human skin tissue samples prepared by the gas-liquid interface contact culture method in Example 6 and the suspension culture method in Example 1 are as follows.

[0027] Figure 8 Example 6 shows the transepithelial resistivity measurements of three-dimensional human epidermal organoids prepared using the gas-liquid interface contact culture method at different time points; where ALI represents the gas-liquid interface contact culture method. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The Ham's F-12 medium used in this embodiment of the invention is a commercially available medium, manufactured by ThermoFisher, catalog number 31765035. This basal medium contains the following components: Amino acids: including glycine 7.5 mg / L, L-alanine 8.9 mg / L, L-arginine hydrochloride 211.0 mg / L, L-asparagine-HO 15.01 mg / L, L-aspartic acid 13.3 mg / L, L-cysteine ​​hydrochloride-HO 35.12 mg / L, L-glutamic acid 14.7 mg / L, L-glutamine 146.0 mg / L, L-histidine hydrochloride-HO 21.0 mg / L, L-isoleucine 4.0 mg / L, L-leucine 13.1 mg / L, L-lysine hydrochloride 36.5 mg / L, L-methionine 4.5 mg / L, L-phenylalanine 5.0 mg / L, L-proline 34.5 mg / L, L-serine 10.5 mg / L, and L-threonine 11.9 mg / L. mg / L, L-tryptophan 2.04 mg / L, L-tyrosine disodium salt dihydrate 7.81 mg / L, L-valine 11.7 mg / L.

[0030] Vitamins: including biotin 0.0073 mg / L, choline chloride 14.0 mg / L, D-calcium pantothenate 0.5 mg / L, folic acid 1.3 mg / L, inositol 18.0 mg / L, nicotinamide 0.036 mg / L, pyridoxine hydrochloride 0.06 mg / L, riboflavin 0.037 mg / L, thiamine hydrochloride 0.3 mg / L, and vitamin B12 1.4 mg / L.

[0031] Inorganic salts: including calcium chloride (CaCl2, anhydrous) 33.22 mg / L, copper sulfate (CuSO4·5H2O) 0.0025 mg / L, ferrous sulfate (FeSO4·7H2O) 0.834 mg / L, magnesium chloride (anhydrous) 57.22 mg / L, potassium chloride (KCl) 223.6 mg / L, sodium bicarbonate (NaHCO3) 1176.0 mg / L, sodium chloride (NaCl) 7599.0 mg / L, disodium hydrogen phosphate (Na2HPO4, anhydrous) 142.0 mg / L, and zinc sulfate (ZnSO4·7H2O) 0.863 mg / L.

[0032] Other components include: D-glucose (glucose) 1802.0 mg / L, sodium hypoxanthine 4.77 mg / L, linoleic acid 0.084 mg / L, lipoic acid 0.21 mg / L, phenol red 1.2 mg / L, putrescine·2HCl 0.161 mg / L, sodium pyruvate 110.0 mg / L, and thymidine 0.7 mg / L.

[0033] Example 1 (a) Preparation of stock solutions for each component in the culture medium The following stock solutions were prepared using Ham's F-12 as a solvent: L-glutamine was dissolved in Ham's F-12 to prepare a 200 mM stock solution.

[0034] Adenine was dissolved in Ham's F-12 to prepare a 4 mM stock solution.

[0035] Epidermal growth factor was dissolved in Ham's F-12 to prepare a stock solution of 50 μg / mL.

[0036] Insulin was dissolved in Ham's F-12 to prepare a stock solution of 1 mg / mL.

[0037] Transferrin was dissolved in Ham's F-12 to prepare a stock solution of 1 mg / mL.

[0038] Calcium chloride was dissolved in Ham's F-12 to prepare a 180 mM stock solution.

[0039] Alanine was dissolved in Ham's F-12 to prepare a stock solution of 890 μg / mL.

[0040] Asparagine was dissolved in Ham's F-12 to prepare a stock solution of 1.33 mg / mL.

[0041] Glutamic acid was dissolved in Ham's F-12 to prepare a stock solution of 1.47 mg / mL.

[0042] Glycine was dissolved in Ham's F-12 to prepare a stock solution of 750 μg / mL.

[0043] Proline was dissolved in Ham's F-12 to prepare a stock solution of 1.15 mg / mL.

[0044] Serine was dissolved in Ham's F-12 to prepare a stock solution of 1.05 mg / mL.

[0045] Arginine was dissolved in Ham's F-12 to prepare a stock solution of 2.1 mg / mL.

[0046] N-acetyl-L-cysteine ​​was dissolved in Ham's F-12 to prepare a 100 mM stock solution.

[0047] β-Mercaptoethanol was dissolved in Ham's F-12 to prepare a 5 mM stock solution.

[0048] The following mother liquors were prepared using DMSO as the solvent: Hydrocortisone was dissolved in DMSO to prepare a 1 mM stock solution.

[0049] Y-27632 was dissolved in DMSO to prepare a 5 mM stock solution.

[0050] A83-01 was dissolved in DMSO to prepare a 10mM stock solution.

[0051] Triiodothyronine was dissolved in DMSO to prepare a 2 μM stock solution.

[0052] Progesterone was dissolved in DMSO to prepare a 200 μM stock solution.

[0053] Linoleic acid was dissolved in DMSO to prepare a stock solution of 50 mg / mL.

[0054] Linolenic acid was dissolved in DMSO to prepare a stock solution of 50 mg / mL.

[0055] Each component of the above culture medium is prepared separately as a stock solution, which can effectively reduce batch-to-batch variability.

[0056] (ii) Preparation of differentiation culture medium Add the stock solutions from step (I) sequentially to Ham's F-12 to prepare the final differentiation medium. The final concentrations of the components in the final differentiation medium are as follows: L-Glutamine (4.8 mM), Adenine (48 μM), Hydrocortisone (1.2 μM), Epidermal Growth Factor (24 ng / mL), Y-27632 (5 μM), A83-01 (5 μM), Triiodothyronine (24 nM), Insulin (12 μg / mL), Transferrin (12 μg / mL), Progesterone (2.4 nM), Calcium Chloride (2.16 mM), Linoleic Acid (0.6 mg / mL), Linolenic Acid (0.6 mg / mL), Alanine (10.68 μg / mL), Asparagine (15.96 μg / mL), Glutamate (17.64 μg / mL), Glycine (9.0 μg / mL), Proline (13.8 μg / mL), Serine (12.6 μg / mL), Arginine (25.2 μg / mL), N-acetyl-L-cysteine ​​(1.2 ng / mL) mM), β-mercaptoethanol (60 μM).

[0057] (III) Construction of three-dimensional human epidermal organoids by suspension culture method (1) Cell seeding and clustering: 5×10 4 Individual adult epidermal stem cells were seeded into low-absorption U-bottom 96-well plates, and 200 μL of commercially available Eplife (Gibco, MEPI500CA) amplification medium was added to each well. The cells were cultured for 24 hours to allow them to form cell clusters.

[0058] (2) Dynamic culture system expansion culture: The cell clusters formed in step (1) are transferred to a 6 cm ultra-low adsorption culture dish. 3 mL of commercially available Eplife expansion medium is added to the culture dish, and the culture is carried out at 55 rpm for 3 days by controlling the shaking speed. The culture medium is changed regularly to achieve large-scale, uniform production and maturation of organoids.

[0059] (3) Induction of differentiation: After suspension culture, the culture medium was replaced with the differentiation medium prepared in step (ii) of Example 1. Differentiation culture was carried out for 10 days with the medium changed every other day to initiate and promote the further development and differentiation of epidermal organoids. Figure 1 The morphology of the three-dimensional human epidermal organoids cultured using the above method is shown 24 hours after construction.

[0060] Results analysis: such as Figure 1 As shown, the epidermal organoid has formed a dense, well-defined spherical aggregate with an intact overall morphology and no obvious central necrosis area, indicating that it has good cell activity and an intact structure.

[0061] Example 2 (a) Preparation of stock solutions for each component in the culture medium Consistent with Example 1 (ii) Preparation of differentiation culture medium Add the stock solutions from step (I) to Ham's F-12 in sequence to prepare the final differentiation medium. The components of the final differentiation medium are as follows: L-Glutamine (2.4 mM), adenine (24 μM), hydrocortisone (0.6 μM), epidermal growth factor (12 ng / mL), Y-27632 (5 μM), A83-01 (10 μM), triiodothyronine (12 nM), insulin (6 μg / mL), transferrin (6 μg / mL), progesterone (1.2 nM), calcium chloride (1.08 mM), linoleic acid (0.3 mg / mL), linolenic acid (0.3 mg / mL), alanine (5.34 μg / mL), asparagine (7.98 μg / mL), glutamate (8.82 ng / mL). μg / mL), glycine (4.5 μg / mL), proline (6.9 μg / mL), serine (6.3 μg / mL), arginine (12.6 μg / mL), N-acetyl-L-cysteine ​​(0.6 mM), β-mercaptoethanol (30 μM).

[0062] (III) Construction of human epidermal organoids by suspension culture (1) Cell seeding and clustering: 2×10 4 Individual adult epidermal stem cells were seeded into low-absorption U-bottom 96-well plates, with 200 μL of commercially available Eplife amplification medium added to each well, and cultured for 24 hours to allow them to form cell clusters.

[0063] (2) Dynamic culture system expansion culture: The cell clusters formed in step (1) are transferred to a 6 cm ultra-low adsorption culture dish. 3 mL of commercially available Eplife expansion medium is added to the culture dish, and the culture is carried out at 95 rpm for 3 days by controlling the shaking speed. The culture medium is changed regularly to achieve large-scale, uniform production and maturation of organoids.

[0064] (4) Induction of differentiation: After suspension culture, the culture medium was replaced with the differentiation medium prepared in step (ii) of Example 2, and cultured for 14 days with the medium changed every other day to initiate and promote the further development and differentiation of epidermal organoids. Figure 2 This shows the morphology of the three-dimensional human epidermal organoids cultured using this method 14 days after differentiation.

[0065] Results analysis: such as Figure 2 As shown, this epidermal organoid exhibits a dense three-dimensional spherical structure with obvious tissue layering and a central cavity morphology, indicating that it has good structural integrity and cell activity.

[0066] To investigate the layered structure of the three-dimensional human epidermal organoids prepared by the method in Example 2, immunohistochemical detection experiments were performed. The specific experimental procedure is as follows: A: Sample fixation and embedding: The cultured mature epidermal organoids were fixed with 4% paraformaldehyde for 24 hours, washed with PBS, and then dehydrated, cleared and embedded in paraffin to form a paraffin block that can be used for sectioning.

[0067] B: Slice preparation: Prepare continuous slices with a thickness of 4-6 μm.

[0068] C: Pre-staining treatment (dewaxing to blocking): After dewaxing and hydration, the sections are subjected to antigen retrieval in citrate buffer (pH 6.0) and allowed to cool naturally to room temperature. Then, endogenous peroxidase activity is blocked with 3% hydrogen peroxide solution, followed by incubation with 5% bovine serum albumin to block non-specific binding sites and reduce non-specific antibody adsorption.

[0069] D: Immunostaining: Specific primary antibodies against KRT5, KRT14, Loricrin, and Filaggrin were added to the slides and incubated overnight at 4°C to allow the antibodies to fully bind to the target antigens. Unbound primary antibodies were washed away with PBS buffer. The corresponding HRP-labeled secondary antibodies were added and incubated at room temperature for 1 hour to allow the secondary antibodies to bind to the primary antibodies.

[0070] E: Staining and Counterstaining: DAB staining solution is added, and under the catalysis of HRP enzyme, a brown precipitate will be produced at the location of the target antigen; Hematoxylin Counterstaining: Hematoxylin is used to counterstain the cell nucleus, making the cell nucleus appear blue, thereby providing a background contrast for the tissue structure.

[0071] F: Mounting and Observation: The sections were dehydrated again with gradient alcohol and cleared with xylene, then mounted with neutral resin for long-term preservation. The prepared sections were observed under an optical microscope. Based on the distribution of the brownish-red (DAB signal), the differentiation characteristics of each layer of the epidermal organoids (basal layer, stratum corneum) were analyzed. The experimental results are as follows: Figure 3 As shown.

[0072] Results analysis: Figure 3 The results show that immunohistochemical detection confirmed the formation of a complete epidermal layer structure, including KRT5 and KRT14 positive basal cells and an outermost keratinized capsule labeled with Loricrin and Filaggrin. These results indicate that the differentiation medium provided by this invention can effectively support the in vitro construction and differentiation maturation of epidermal organoids with a complete layer structure.

[0073] Example 3 (a) Preparation of stock solutions for each component in the culture medium Consistent with Example 1 (ii) Preparation of differentiation culture medium Add the stock solutions from step (I) to Ham's F-12 in sequence to prepare the final differentiation medium. The components of the final differentiation medium are as follows: L-Glutamine (9.6 mM), Adenine (96 μM), Hydrocortisone (2.4 μM), Epidermal Growth Factor (48 ng / mL), Y-27632 (2 μM), A83-01 (5 μM), Triiodothyronine (48 nM), Insulin (24 μg / mL), Transferrin (24 μg / mL), Progesterone (4.8 nM), Calcium Chloride (1.2 mM), Linoleic Acid (1.2 mg / mL), Linolenic Acid (1.2 mg / mL), Alanine (21.36 μg / mL), Asparagine (31.92 μg / mL), Glutamate (35.28 μg / mL), Glycine (18.0 μg / mL), Proline (27.6 μg / mL), Serine (25.2 μg / mL), Arginine (50.4 μg / mL), N-acetyl-L-cysteine ​​(2.4 ng / mL) mM), β-mercaptoethanol (120 μM).

[0074] (III) Construction of human epidermal organoids by suspension culture (1) Cell seeding and clustering: 1×10 4 Individual adult epidermal stem cells were seeded into low-absorption U-bottom 96-well plates, with 200 μL of commercially available Eplife amplification medium added to each well, and cultured for 24 hours to allow them to form cell clusters.

[0075] (2) Dynamic culture system expansion culture: The cell clusters formed in step (1) are transferred to a 6 cm ultra-low adsorption culture dish. 3 mL of commercially available Eplife expansion medium is added to the culture dish, and the culture is carried out at 80 rpm for 3 days by controlling the shaking speed. The culture medium is changed regularly to achieve large-scale, uniform production and maturation of organoids.

[0076] (3) Induction of differentiation: After the suspension culture is completed, the culture medium is replaced with the differentiation culture medium prepared in step (II) of Example 3. The culture is carried out for 16 days and the medium is changed every other day to initiate and promote the further development and differentiation of epidermal organoids.

[0077] To investigate the layered structure of the three-dimensional human epidermal organoids prepared by the method in Example 3, immunofluorescence detection of specific biomarkers was performed on the three-dimensional human epidermal organoids cultured by the method in Example 3. The specific experimental procedure is as follows: Organoids induced to differentiate were further cultured on day 3 of cell passage using a gas-liquid interface contact method, and cultured for another 14 days in differentiation-inducing medium to stimulate their multi-lineage differentiation potential. After induction, samples were fixed with 4% paraformaldehyde at 4°C for 30 min, dehydrated with 30% sucrose solution for 24 h, embedded in OCT embedding medium, and cryosectioned. After blocking, specific primary antibodies (KRT5) against epidermal basal layer markers and KRT10 against terminal differentiation markers were added and incubated overnight at 4°C. The sections were then washed three times with PBS for 5 min each time, followed by incubation with corresponding species-derived fluorescently labeled secondary antibodies at room temperature in the dark for 1 h. After three repeated washings with PBS, the nuclei were counterstained with DAPI for 5 min, and finally washed three more times with PBS. Images were then observed and acquired under a fluorescence microscope. Experimental results are as follows: Figure 4 As shown.

[0078] Results analysis: such as Figure 4 As shown, the epidermal organoids exhibit a distinct spatial partitioning structure: the central region expresses the keratinocyte terminal differentiation marker (KRT10), while the peripheral region expresses the basal cell marker (KRT5). This clear differentiation gradient confirms that the culture medium of this invention can effectively guide the terminal differentiation process of epidermal organoids and promote the formation of their functional keratinocyte capsule.

[0079] Example 4 (a) Preparation of stock solutions for each component in the culture medium Consistent with Example 1 (ii) Preparation of differentiation culture medium Add the stock solutions from step (I) to Ham's F-12 in sequence to prepare the final differentiation medium. The components of the final differentiation medium are as follows: L-Glutamine (8 mM), Adenine (50 μM), Hydrocortisone (3 μM), Epidermal Growth Factor (40 ng / mL), Y-27632 (2 μM), A83-01 (10 μM), Triiodothyronine (40 nM), Insulin (20 μg / mL), Transferrin (20 μg / mL), Progesterone (4 nM), Calcium Chloride (1.5 mM), Linoleic Acid (1.08 mg / mL), Linolenic Acid (1.08 mg / mL), Alanine (19.224 μg / mL), Asparagine (28.728 μg / mL), Glutamate (31.752 μg / mL), Glycine (16.2 μg / mL), Proline (24.84 μg / mL), Serine (22.68 μg / mL), Arginine (45.36 μg / mL), N-acetyl-L-cysteine ​​(2.16 μg / mL) mM), β-mercaptoethanol (108 μM).

[0080] (III) Construction of human epidermal organoids by gas-liquid interface contact culture method (1) Construction of a cell-free basal layer: In the Transwell chamber, type I rat tail collagen was neutralized to neutral with 0.1M sodium hydroxide solution and subjected to three-dimensional gelation to form a cell-free basal layer.

[0081] (2) Establishment of a dermal model: 50 μL of human dermal fibroblasts were mixed with 300 μL of type I rat tail collagen, and then... 4 Cells / cm² were seeded at a density corresponding to the number of nuclei on the cell-free basal layer to construct the dermis, and cultured in DMEM medium containing 10% FBS.

[0082] (3) Dermal layer pretreatment: The system obtained in step (2) was cultured for 5 days, then the DMEM medium containing 10% FBS was removed and the system was allowed to stand for 15 minutes under sterile conditions.

[0083] (4) Inoculation with human adult epidermal stem cells: at a dose of 5 × 10⁻⁶ 4 Human adult epidermal stem cells were collected at a density of cells / cm², centrifuged, and resuspended in 20 μL of commercially available Eplife amplification medium. They were then evenly seeded onto the dermal surface of step (3), left to stand for 15 minutes, and then placed in an incubator for 30 minutes. Subsequently, commercially available Eplife amplification medium was added and cultured for 3 days.

[0084] (5) Gas-liquid interface induced differentiation: Add the differentiation medium from step (ii) of Example 4 to the lower chamber of Transwell, and do not add medium to the upper chamber to establish a gas-liquid interface culture environment; change the medium in the lower chamber every day and continue to culture for 20 days to induce the formation and differentiation of the epidermis.

[0085] To investigate the properties of the three-dimensional human epidermal organoids prepared by the method in Example 4, immunofluorescence detection using specific biomarkers was performed. The specific experimental procedure is as follows: Organoids induced to differentiate were further cultured on day 3 of cell passage using a gas-liquid interface contact method, and cultured for another 20 days in differentiation-inducing medium to stimulate their multi-lineage differentiation potential. After induction, samples were fixed with 4% paraformaldehyde at 4°C for 30 min, dehydrated with 30% sucrose solution for 24 h, embedded in OCT embedding medium, and cryosectioned. After blocking, specific primary antibodies (KRT5) against epidermal basal layer markers and KRT10 against terminal differentiation markers were added and incubated overnight at 4°C. The sections were then washed three times with PBS for 5 min each time, followed by incubation with corresponding species-derived fluorescently labeled secondary antibodies at room temperature in the dark for 1 h. After three repeated washings with PBS, the nuclei were counterstained with DAPI for 5 min, and finally washed three more times with PBS. Images were then observed and acquired under a fluorescence microscope. Figure 5 The results of immunofluorescence staining of three-dimensional human epidermal organoids cultured using the method of Example 4 of this invention at different culture time points are shown.

[0086] Results Analysis: Figure 5 It is evident that the differentiation culture medium and method provided by this invention can effectively induce the in vitro construction and differentiation maturation of physiologically relevant human epidermal organoids. Including basal cells (KRT5) and terminally differentiated epidermal keratinocytes (KRT10), it is confirmed that their excellent multi-lineage differentiation potential can be maintained for a long time, with tight intercellular connections and a tissue structure highly similar to the characteristics of adult skin epidermis.

[0087] Example 5 (a) Preparation of stock solutions for each component in the culture medium Consistent with that in Example 1.

[0088] (ii) Preparation of culture medium Add the stock solutions from step (I) sequentially to Ham's F-12 to prepare the final culture medium. The components of the final culture medium are as follows: L-Glutamine (4 mM), Adenine (40 μM), Hydrocortisone (1 μM), Epidermal Growth Factor (20 ng / mL), Y-27632 (5 μM), A83-01 (10 μM), Triiodothyronine (20 nM), Insulin (10 μg / mL), Transferrin (10 μg / mL), Progesterone (2 nM), Calcium Chloride (1.8 mM), Linoleic Acid (0.5 mg / mL), Linolenic Acid (0.5 mg / mL), Alanine (8.9 μg / mL), Asparagine (13.3 μg / mL), Glutamic Acid (14.7 μg / mL), Glycine (7.5 μg / mL), Proline (11.5 μg / mL), Serine (10.5 μg / mL), Arginine (21 μg / mL), N-Acetyl-L-cysteine ​​(1 mM), β-Mercaptoethanol (50 μM).

[0089] (III) Construction of human epidermal organoids by gas-liquid interface contact culture method (1) Construction of a cell-free basal layer: In the Transwell chamber, type I rat tail collagen was neutralized to neutral with 0.1M sodium hydroxide solution and subjected to three-dimensional gelation to form a cell-free basal layer.

[0090] (2) Establishment of a dermal model: 50 μL of human dermal fibroblasts were mixed with 300 μL of type I rat tail collagen at a concentration of 2 × 10⁻⁶. 4 Cells / cm² were seeded onto the cell-free basal layer to construct the dermis, and cultured in DMEM medium containing 10% FBS.

[0091] (3) Dermal layer pretreatment: The system obtained in step (2) was cultured for 5 days, then the DMEM medium containing 10% FBS was removed and the system was allowed to stand for 15 minutes under sterile conditions.

[0092] (4) Inoculation with human adult epidermal stem cells: at 6.0 × 10⁻⁶ 4 Human adult epidermal stem cells were collected at a density of cells / cm², centrifuged, and resuspended in 20 μL of commercially available Eplife amplification medium. They were then evenly seeded onto the dermal surface of step (3), left to stand for 15 minutes, and then placed in an incubator for 30 minutes. Subsequently, commercially available Eplife amplification medium was added and cultured for 3 days.

[0093] (5) Gas-liquid interface induced differentiation: Add the differentiation medium prepared in step (ii) of Example 5 to the lower chamber of Transwell, and do not add medium to the upper chamber to establish a gas-liquid interface culture environment; change the medium in the lower chamber every day and continue to culture for 14 days to induce the formation and differentiation of the epidermis.

[0094] To investigate the properties of the three-dimensional human epidermal organoids prepared by the method in Example 5, immunofluorescence detection using specific biomarkers was performed. The specific experimental procedure is as follows: Organoids induced to differentiate were further cultured on day 3 of cell passage using a gas-liquid interface contact method, and cultured for another 14 days in differentiation-inducing medium to stimulate their multi-lineage differentiation potential. After induction, samples were fixed with 4% paraformaldehyde at 4°C for 30 min, dehydrated with 30% sucrose solution for 24 h, embedded in OCT embedding medium, and cryosectioned. After blocking, specific primary antibodies (including KRT10, Loricrin, and Filaggrin) against epidermal basal layer markers (KRT5) and terminal differentiation markers were added and incubated overnight at 4°C. The sections were then washed three times with PBS for 5 min each time, followed by incubation with corresponding species-derived fluorescently labeled secondary antibodies at room temperature in the dark for 1 h. After three washes with PBS, the nuclei were counterstained with DAPI for 5 min, and finally washed three more times with PBS. Images were then observed and acquired under a fluorescence microscope. Figure 6 The results of immunofluorescence staining of three-dimensional human epidermal organoids cultured using the method of Example 5 of this invention are shown at 14 days after induced differentiation.

[0095] Results Analysis: Specific marker detection revealed a complete epidermal differentiation lineage: basal cells (KRT5 positive), spinous and stratum corneum (KRT10 positive), and granular layer (Loricrin and Filaggrin positive) structures. This epidermal organoid exhibited tight cell junctions and clear spatial stratification, with its tissue structure highly consistent with adult skin epidermal characteristics. These results demonstrate that the culture medium and method provided by this invention can effectively support the in vitro construction and complete differentiation process of physiologically relevant human epidermal organoids.

[0096] Example 6 (a) Preparation of stock solutions for each component in the culture medium Consistent with Example 1.

[0097] (ii) Preparation of culture medium Consistent with Example 5.

[0098] (III) Construction of human epidermal organoids by gas-liquid interface contact culture method (1) Construction of a cell-free basal layer: In the Transwell chamber, type I rat tail collagen was neutralized to neutral with 0.1M sodium hydroxide solution and subjected to three-dimensional gelation to form a cell-free basal layer.

[0099] (2) Establishment of a dermal model: 50 μL of human dermal fibroblasts were mixed with 300 μL of type I rat tail collagen, and then... 4The cells / cm² were seeded onto the basal layer to construct the dermis, and then cultured in DMEM medium containing 10% FBS.

[0100] (3) Dermal layer pretreatment: The system obtained in step (2) was cultured for 5 days, then the DMEM medium containing 10% FBS was removed and the system was allowed to stand for 15 minutes under sterile conditions.

[0101] (4) Inoculation with human adult epidermal stem cells: at 8.0 × 10⁻⁶ 4 Human adult epidermal stem cells were collected at a density of cells / cm², centrifuged, and resuspended in 20 μL of commercially available Eplife amplification medium. They were then evenly seeded onto the dermal surface of step (3), left to stand for 15 minutes, and then placed in an incubator for 30 minutes. Subsequently, commercially available Eplife amplification medium was added and cultured for 3 days.

[0102] (5) Gas-liquid interface induced differentiation: Add the differentiation medium prepared in step (ii) of Example 6 to the lower chamber of Transwell, and do not add medium to the upper chamber to establish a gas-liquid interface culture environment; change the medium in the lower chamber every day and continue to culture for 14 days to induce the formation and differentiation of the epidermis.

[0103] To investigate the properties of the three-dimensional human epidermal organoids prepared by the method in Example 6, they were stained with hematoxylin-eosin (HE). The specific experimental procedure is as follows: (1) Sample fixation and pretreatment: The three-dimensional human epidermal organoids cultured into heat were fixed at 4°C for 30 minutes using 4% paraformaldehyde solution. After being thoroughly rinsed with PBS, they were dehydrated at 4°C for 24 hours using 30% sucrose solution.

[0104] (2) Embedding and sectioning: The dehydrated sample was embedded with OCT embedding agent, and continuous sections with a thickness of 6-8 μm were prepared in a cryostat and attached to a glass slide treated with polylysine.

[0105] (3) Staining treatment Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5-8 minutes, rinse with running tap water, differentiate with 1% hydrochloric acid ethanol for a few seconds, and then rinse with running water for 5 minutes to return to blue.

[0106] Eosin staining: The sections stained with hematoxylin were dehydrated in 70%, 85%, and 95% ethanol gradients for 1 minute each, and then immersed in eosin staining solution for 30 seconds.

[0107] (4) Dehydration and mounting: The stained sections were rinsed twice with 95% ethanol and anhydrous ethanol, each time for 30 seconds, then cleared with xylene for 5 minutes, and finally mounted with neutral resin.

[0108] (5) Microscopic observation: The prepared sections are placed under an optical microscope for observation to evaluate the layered structure, cell morphology and tissue characteristics of the epidermal organoids. Figure 7 The results of hematoxylin-eosin (HE) staining comparison of human epidermal organoids prepared using Examples 6 and 1 of the present invention are shown.

[0109] Results analysis: such as Figure 7 As shown, the epidermal organoids constructed by the gas-liquid interface culture method and the suspension culture method both exhibited tissue structure characteristics similar to human skin, including complete epidermal layer structure and cell morphology characteristics, which confirms the effectiveness of the culture system of the present invention in simulating human skin tissue structure.

[0110] Figure 8 The results of transepithelial resistivity measurements of human epidermal organoids cultured using the method of step (iii) of Embodiment 6 of the present invention are shown at different time points.

[0111] Results analysis: As shown in the figure, with the extension of the induced differentiation time, the transepithelial resistance value of the epidermal organoids showed a continuous upward trend, eventually reaching about 680 Ω·cm², indicating that its barrier function gradually improved and reached the typical characteristics of mature epidermal tissue.

[0112] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0113] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A differentiation culture medium for three-dimensional human epidermal organoids, characterized in that, The differentiation medium consisted of Ham's F-12 basal medium and the following components at final concentrations: 2.4-9.6 mM L-glutamine, 24-96 μM adenine, 0.6-3 μM hydrocortisone, 12-48 ng / mL epidermal growth factor, 2-5 μM Y-27632, 5-10 μM A83-01, 12-48 nM triiodothyronine, 6-24 μg / mL insulin, 6-24 μg / mL transferrin, 1.2-4.8 nM progesterone, 1.08-2.16 mM calcium chloride, 0.3-1.2 mg / mL linoleic acid, and 0.3-1.2 mg / mL linoleic acid. mg / mL linolenic acid, 5.34-21.36 μg / mL alanine, 7.98-31.92 μg / mL asparagine, 8.82-35.28 μg / mL glutamate, 4.5-18.0 μg / mL glycine, 6.9-27.6 μg / mL proline, 6.3-25.2 μg / mL serine, 12.6-50.4 μg / mL arginine, 0.6-2.4 mM N-acetyl-L-cysteine, and 30-120 μM β-mercaptoethanol.

2. The differentiation culture medium according to claim 1, characterized in that, The differentiation medium consisted of Ham's F-12 basal medium and the following components at final concentrations: 4 mM L-glutamine, 40 μM adenine, 1 μM hydrocortisone, 20 ng / mL epidermal growth factor, 5 μM Y-27632, 5 μM A83-01, 20 nM triiodothyronine, 10 μg / mL insulin, 10 μg / mL transferrin, 2 nM progesterone, 1.8 mM calcium chloride, 0.5 mg / mL linoleic acid, 0.5 mg / mL linolenic acid, 8.9 μg / mL alanine, 13.3 μg / mL asparagine, 14.7 μg / mL glutamate, 7.5 μg / mL glycine, 11.5 μg / mL proline, 10.5 μg / mL serine, 21 μg / mL arginine, 1 mM N-acetyl-L-cysteine, and 50 μM β-mercaptoethanol.

3. The method for preparing the differentiation culture medium according to claim 1 or 2, characterized in that, Includes the following steps: (1) L-glutamine, adenine, epidermal growth factor, insulin, transferrin, calcium chloride, alanine, asparagine, glutamic acid, glycine, proline, serine, arginine, N-acetyl-L-cysteine ​​and β-mercaptoethanol were dissolved in Ham's F-12 to prepare corresponding stock solutions. (2) Dissolve hydrocortisone, A83-01, Y-27632, triiodothyronine, progesterone, linoleic acid and linolenic acid in DMSO to prepare corresponding stock solutions; (3) Add each mother liquor from steps (1) to (2) to Ham's F-12 according to the final concentration of each component in the culture medium to prepare differentiation culture medium.

4. The application of the differentiation culture medium according to any one of claims 1-2 or the differentiation culture medium prepared by the method of claim 3, characterized in that, The application is in any of the following directions: (1) Application in three-dimensional human epidermal organoid differentiation culture; (2) Application in the preparation of a three-dimensional human epidermal organoid differentiation culture kit; (3) Application in the preparation of three-dimensional human epidermal organoid in vitro models; (4) Application in in vitro screening and evaluation of cosmetics and toxic drugs.

5. A kit for three-dimensional human epidermal organoid differentiation culture, characterized in that, The differentiation culture medium includes the one described in any one of claims 1-2.

6. A method for culturing three-dimensional human epidermal organoids, characterized in that, The process includes the following steps: using the differentiation culture medium described in claim 1 or 2 as a basis, human adult epidermal stem cells are induced to differentiate and cultured by gas-liquid interface contact culture or suspension culture to prepare three-dimensional human epidermal organoids.

7. The cultivation method according to claim 6, characterized in that, The specific process of the gas-liquid interface contact culture method is as follows: (a) Construction of a cell-free basal layer: In a Transwell chamber, type I rat tail collagen was neutralized to neutral using 0.1M sodium hydroxide solution for three-dimensional gelation to form a cell-free basal layer; (b) Establishment and pretreatment of the dermis: 50 μL of human dermal fibroblasts were mixed with 300 μL of type I rat tail collagen and then subjected to a reaction at a concentration of 1.5–2.5 × 10⁻⁶. 4 The cells / cm² were inoculated onto the cell-free basal layer. After culturing in culture medium for 3-5 days, the culture medium was removed and the mixture was allowed to stand for 15 minutes under sterile conditions. (c) Inoculation with human adult epidermal stem cells: Human adult epidermal stem cells were inoculated at a rate of 5-8 × 10⁻⁶. 4 The cells / cm² were seeded on the surface of the dermal layer obtained in step (b), left to stand for 15 min, and then placed in an incubator for 30 min. After that, they were cultured in amplification medium for 3-5 days. (d) Gas-liquid interface culture to induce differentiation: Add the differentiation medium according to any one of claims 1-2 to the lower chamber of the Transwell, while no medium is added to the upper chamber to establish a gas-liquid interface culture environment. Change the medium every day and continue culturing to induce the epidermis to form a complete layered structure. The specific process of the suspension culture method is as follows: (i) Divide 1-5×10 4 Individual adult epidermal stem cells were seeded in low-absorption multi-well plates and cultured using expansion medium to form cell clusters. (ii) Transfer the cell clusters formed in step (i) to the amplification medium for shaker suspension amplification culture; (iii) After the culture in step (ii) is completed, the culture medium is replaced with the differentiation culture medium described in any one of claims 1-2, and the suspension culture is continued to induce the differentiation and maturation of epidermal organoids.

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