A method for constructing a recombinant human vaginal full-thickness model in vitro, products and applications

Abstract

This invention, entitled "A Method for In Vitro Construction of a Recombinant Human Vaginal Full-Thickness Model, its Product, and its Application," belongs to the field of biomedical technology. The technical problem to be solved is to provide an in vitro construction method for a recombinant human vaginal full-thickness model to improve issues such as unreasonable cell sources, poor physiological relevance of the model, and insufficient stability. The key technical points are as follows: The in vitro construction method for the recombinant human vaginal full-thickness model includes: S1, mixing and culturing human skin fibroblasts with matrix material to obtain a matrix gel, and continuing to culture to form the lamina propria; S2, seeding VK2 / E6E7 cells onto the surface of the lamina propria; S3, using a gas-liquid interface culture method to induce differentiation and culture of the VK2 / E6E7 cells seeded on the surface of the lamina propria to form an epithelial layer, thereby obtaining a recombinant human vaginal full-thickness model.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the field of biological model technology, and relates to an in vitro construction method, product, and application of a recombinant human vaginal full-thickness model. Background Technology

[0002] As a vital barrier of the female reproductive system, the integrity of the vagina's structure and function is crucial for maintaining reproductive health. In vitro constructed full-thickness models of the vagina can simulate the physiological structure and microenvironment of the human vagina, possessing irreplaceable application value in fields such as gynecological disease pathogenesis research, drug screening and toxicity evaluation, and the development of mucosal repair materials.

[0003] Currently, the core idea behind constructing a full-thickness vaginal model in vitro is to replicate the layered structure of the human vagina, namely the epithelial layer composed of epithelial cells and the lamina propria composed of fibroblasts and other cells. In existing technologies, the cell sources used to construct this model mainly fall into two categories: one is a combination of tumor cell lines (such as A431 cells) and primary vaginal fibroblasts; the other is a combination of primary vaginal epithelial cells and primary vaginal fibroblasts. A431 cells are an immortalized cell line derived from human epidermal squamous cell carcinoma, and due to their ease of culture and strong proliferative capacity, they have been used in some studies to replace epithelial cells in constructing mucosal models. Primary vaginal epithelial cells and primary vaginal fibroblasts are directly derived from human vaginal tissue, theoretically more closely resembling the physiological state, but their acquisition and culture face certain limitations.

[0004] The basic process of existing construction techniques is as follows: first, fibroblasts are seeded in a matrix material (such as collagen gel, Matrigel, etc.) and cultured to form a lamina propria scaffold; then, epithelial cells are seeded on top; and epithelial cell differentiation is induced by adjusting the culture environment (such as gas concentration, nutrient composition, and culture time), ultimately forming a full-thickness vaginal model with a layered structure. These techniques provide basic tools for vaginal-related in vitro research, but there is still room for improvement in terms of model stability, physiological similarity, and reproducibility.

[0005] Relevant patent documents retrieved: The publication country is China, publication number CN113293126A, publication date August 24, 2021, entitled "An In Vitro Construction Method for a Human Vaginal Mucosal Model." This document discloses an in vitro construction method for a human vaginal mucosal model. A matrix layer is constructed using vaginal fibroblasts and collagen, upon which vaginal epithelial cells are inoculated to form an in vitro vaginal mucosal model with a double-layered structure. Structurally, it is highly similar to natural tissue. Vaginal irritation experiments conducted on this model closely resemble the actual in vivo situation, and the experimental results are accurate and reliable. The co-culture of vaginal epithelial cells and the matrix layer employs two stages: submerged culture and gas-liquid surface culture. Furthermore, the nutrient composition of the culture medium is finely adjusted at different stages to meet the varying needs of cell proliferation and differentiation. This segmented culture method ensures the nutritional needs of cells at different developmental stages, facilitating the formation of a multilayered vaginal mucosal epithelial structure, while also shortening the construction time and reducing production costs.

[0006] Relevant non-patent literature retrieved: The document, titled "Experimental Study on Construction of Tissue-Engineered Vaginal Model by Rabbit Bone Marrow Mesenchymal Stem Cells Composite with Small Intestinal Submucosal Layer (SIS)," published on May 1, 2013, by Peng Qiang of Nanchang University, discloses that BMSC-induced cells, as seed cells, can be composited with SIS material and cultured to continue to proliferate and differentiate in vivo, forming a multilayered cell structure that can be used to construct a tissue-engineered vagina.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) Insufficient rationality of cell source and poor physiological similarity of model: The A431 cells used in patent document CN113293126A are derived from the epidermis rather than the vaginal mucosa. Their morphology, differentiation characteristics, and physiological functions are significantly different from normal vaginal epithelial cells, which makes it impossible for the model to truly simulate the key physiological characteristics of human vaginal epithelium, such as barrier function and secretory function. If primary vaginal epithelial cells are used, there are problems such as scarce source, many ethical restrictions, weak in vitro proliferation capacity, and limited passage times, making it difficult to achieve large-scale and standardized preparation of the model.

[0008] (2) Limited selection of fibroblasts and poor model stability: The existing technology uses primary human vaginal fibroblasts to construct the lamina propria, which also has problems such as limited sources, easy aging in vitro, and large differences in cell characteristics from different donor sources. This leads to poor consistency of models constructed from different batches, making it difficult to guarantee experimental reproducibility, and phenotypic changes are likely to occur during long-term in vitro culture, affecting the long-term stability of the model.

[0009] (3) The model tissue structure has low similarity to the human vagina: The epithelial cells in the model constructed by the existing technology are not fully differentiated, making it difficult to form the complete stratified squamous epithelial structure unique to the human vagina (such as clear stratification of the basal layer, spinous layer and granular layer), and the connection between the epithelial cells and the lamina propria (such as hemidesmosomes and basement membrane) is not tight enough, making it impossible to perfectly reproduce the complete barrier structure of the human vaginal mucosa.

[0010] (4) Poor practicality of the preparation process and difficulty in large-scale application: The construction method based on primary cells has problems such as complex cell separation and culture process and long time consumption. Moreover, the scarcity of primary cells leads to high model preparation cost. Although the construction method based on A431 cells has reduced the difficulty of cell culture, it cannot meet the needs of precise research due to poor physiological correlation of the model, which limits its practical application scenarios.

[0011] In summary, although the technological approach has continuously evolved from stem cell-animal-in vivo construction to primary cell-human-in vitro construction, it has consistently failed to simultaneously resolve the contradiction between "physiological similarity" and "construction stability / scalability" of the model. How to screen for cells that more closely resemble the physiological characteristics of normal human vaginal epithelial cells, select cells with high stability and abundant supply, and optimize culture conditions to construct an in vitro model with fully differentiated epithelial layer, stable lamina propria structure, and tight intercellular connections has become a pressing technical problem to be solved in this field. Summary of the Invention

[0012] The purpose of this invention is to provide: A method for in vitro construction of a recombinant human vaginal full-thickness model, its products and applications, and related technologies, to solve the technical problems, or combinations thereof, in the in vitro construction of vaginal full-thickness models, such as unreasonable cell sources, poor physiological relevance of the model, insufficient stability, low similarity of tissue structure to human tissue, and difficulty in scaling up the preparation process.

[0013] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0014] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0015] Definitions of the standard terminology can be found in the references “Cell Biology (5th Edition), Higher Education Press, authors: Ding Mingxiao, Wang Xizhong, Zhang Chuanmao, Chen Jianguo, et al., 2020”, “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19”, and “Genetic Engineering, Higher Education Press, 2013-08-01”.

[0016] Unless otherwise specified, conventional methods within the scope of the art, such as hematoxylin-eosin (HE) staining, Masson staining, PAS staining, cell viability assay (trypan blue staining), seeding, passage, etc., shall be used.

[0017] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0018] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0019] The term "Transwell" as used in this article refers to a laboratory apparatus (culture device) commonly used for cell co-culture, migration, and invasion experiments. It consists of an independent chamber with a permeable polycarbonate membrane at the bottom, possessing a specific pore size (e.g., 0.4 μm or 8.0 μm). This chamber is placed within a cell culture plate to form an "upper chamber" (inside the transwell) and a "lower chamber" (inside the culture plate). Material exchange between the upper and lower chambers is facilitated by the permeable membrane at the bottom. In tissue engineering model construction, Transwell chambers are primarily used to achieve a "gas-liquid interface culture" mode. Cells are seeded onto the bottom membrane surface of the chamber. Initially, culture medium is added to both chambers to support cell proliferation and fusion. Once a certain cell density is reached, the culture medium in the upper chamber is removed, exposing the apical surface of the cells to air, while the basal surface remains immersed in the culture medium in the lower chamber, thus enabling gas-liquid interface culture.

[0020] The term "VK2 / E6E7 cells" used in this article refers to human vaginal epithelial adherent cells, which are immortalized human vaginal epithelial cell lines derived from human vaginal epithelium. Their morphology, differentiation characteristics, and physiological functions are highly consistent with normal vaginal epithelial cells, and they can be immortalized through culture.

[0021] The term "primary human skin fibroblasts" as used herein refers to fibroblasts derived from human skin tissue (such as neonatal foreskin, adult skin biopsy samples, etc.). These cells are the most abundant type of mesenchymal cell in the dermal connective tissue of the skin, responsible for synthesizing and secreting collagen, elastic fibers, and extracellular matrix components, playing a central role in maintaining skin structural integrity and repairing damage. In this invention, human skin fibroblasts are used to construct the lamina propria (stromal layer) of a reconstructed full-thickness human vaginal model, serving as supporting cells in a three-dimensional scaffold and mimicking the function of fibroblasts in the lamina propria of the human vagina. This term encompasses both primary human skin fibroblasts and immortalized human skin fibroblasts, as defined below.

[0022] The term "primary human skin fibroblasts" as used in this article refers to primary fibroblasts obtained directly from human skin tissue (such as neonatal foreskin or adult skin samples). Compared with primary vaginal fibroblasts, human skin fibroblasts have advantages such as easier access to tissue sources, strong in vitro proliferation capacity, and good passage stability. In tissue engineering, they are often used as supporting cells for constructing dermal equivalents or three-dimensional scaffolds.

[0023] The term "immortalized human skin fibroblasts" as used in this article refers to immortalized cell lines established by genetic engineering techniques (such as transfection with human telomerase reverse transcriptase gene, SV40 large T antigen, or HPV E6 / E7 gene) to grant human skin fibroblasts unlimited proliferative capacity. Unlike primary human skin fibroblasts, which have a limited number of passages and are prone to senescence, immortalized human skin fibroblasts can be cultured in vitro for extended periods and maintain stable proliferative activity and cell phenotype. Common immortalized human skin fibroblast cell lines include, but are not limited to, BJ-hTERT cells and HFF-hTERT cells.

[0024] The term "neutral collagen scaffold" as used in this article refers to an artificial extracellular matrix scaffold used in tissue engineering to provide a three-dimensional framework for cell growth, adhesion, proliferation, and differentiation. It is constructed with collagen as the main component, forming a three-dimensional porous structure (i.e., ultimately adjusted to the physiological pH range). Typically, this is achieved by mixing the matrix material with a cell suspension and forming a gel. In the full-thickness vaginal model, the neutral collagen scaffold serves as a component of the lamina propria (matrix) to load fibroblasts, forming a three-dimensional structure that mimics the lamina propria of the human vagina.

[0025] The term "air-liquid interface culture" used in this article refers to Air-Liquid Interface (ALI), a type of in vitro tissue culture method in which cells are seeded onto a biocompatible membrane (Transwell membrane), with the basal surface of the cells in contact with the culture medium to obtain nutrients, while the apical surface is directly exposed to the air. This culture mode simulates the physiological environment of a large number of epithelial tissues (such as respiratory tract, digestive tract mucosa, vaginal epithelium, etc.) in vivo, namely, the cell layer is located deep inside the tissue, with cells receiving nutrients from body fluids, while the surface layer is exposed to the semi-open environment of organ cavities.

[0026] The term "serum-free keratinocyte culture medium" as used in this article refers to a complete culture medium with a defined chemical composition, free of serum, optimized for the growth and maintenance of human keratinocytes and other types of epithelial cells without the need for a cell feeder layer. For example, Keratinocyte-SFM medium, manufactured by Gibco (product number 17005042), is specifically designed for optimizing the growth and maintenance of human keratinocytes and other types of epithelial cells without the need for a cell feeder layer. This medium is serum-free and used under low calcium ion concentration (<0.1 mM) conditions. When using this medium, recombinant human epidermal growth factor (rEGF) and bovine pituitary extract (BPE) must be added to support the serum-free culture and expansion of keratinocytes.

[0027] The term "EGF" used in this article refers to Epidermal Growth Factor, a heat-stable single-chain low-molecular-weight polypeptide composed of 53 amino acid residues, belonging to the category of cell growth factors. EGF effectively promotes the growth, proliferation, and differentiation of various tissue cells (especially fibroblasts and various epithelial cells) by binding with high affinity to the epidermal growth factor receptor (EGFR) on the surface of target cells, thereby activating a series of intracellular signaling pathways.

[0028] The term "HE staining" used in this article refers to Hematoxylin and Eosin staining, the most commonly used and fundamental staining method in histology and pathology. Its principle is as follows: Hematoxylin is a basic dye that stains the chromatin (DNA) in the cell nucleus blue-purple; eosin is an acidic dye that stains the proteins in the cytoplasm and the extracellular matrix pink to red. HE staining allows for clear differentiation between the cell nucleus and cytoplasm, enabling observation of the general morphology and structure of tissues or cells, cell arrangement, cell layers, and the presence or absence of lesions. In tissue engineering and regenerative medicine research, HE staining is the preferred method for assessing the structural integrity and maturity of in vitro constructed tissue or organ models.

[0029] The term "Masson staining" as used in this article refers to Masson's trichrome staining, a classic three-color staining method used to visualize collagen fibers in tissues. Its principle is based on the selective staining of tissue components using dyes of different molecular sizes: small molecules like aniline blue or brilliant green can penetrate dense collagen fibers and stain them blue or green; while large molecules like acid fuchsin or ponceau red are stained red by muscle fibers, erythrocytes, etc. Masson staining clearly distinguishes collagen fibers (blue / green), muscle fibers and cytoplasm (red), and cell nuclei (dark brown). In tissue engineering, Masson staining is commonly used to assess the distribution, density, and maturity of collagen in the lamina propria or stroma.

[0030] The term "PAS staining" used in this article refers to Periodic Acid-Schiff staining, a special staining method used to detect neutral mucopolysaccharides such as glycogen, mucopolysaccharides, and glycoproteins in tissues or cells. Its principle is as follows: periodic acid oxidizes the ethylene glycol groups in carbohydrates to aldehyde groups, and the aldehyde groups react with Schiff's reagent (colorless fuchsin) to form a purple-red precipitate. A positive PAS reaction (purple-red) indicates the presence of glycogen or glycoprotein. In vaginal epithelial histological evaluation, PAS staining is often used to detect glycogen content in epithelial cells because normal human vaginal stratified squamous epithelium has the function of synthesizing and storing glycogen, and glycogen level is one of the important indicators for evaluating the functional maturity of vaginal epithelium.

[0031] The term "cell pretreatment" as used in this article refers to technical means of improving cell tolerance, functional activity, and therapeutic potential by intervening under specific culture conditions before cell transplantation or subsequent culture.

[0032] The term "cell confluency" used in this article refers to the percentage of cells that adhere to and spread on the bottom surface of the culture vessel (such as a culture dish, culture flask, or multi-well plate) as observed under a microscope during adherent cell culture, relative to the total area that can adhere.

[0033] The term "cell viability" used in this article refers to "cell survival rate" or "cell viability," which is the proportion of live cells with intact cell membrane structure and normal metabolic function in a cell population. This indicator is expressed as a percentage (%) and is a key technical parameter for assessing cell health, proliferation capacity, and experimental suitability. It can be determined using the trypan blue staining method.

[0034] The term "passage culture" used in this article refers to the technique of dividing the culture and re-inoculating it into new culture dishes to continue growth, which is used to solve the problem of growth restriction caused by cell contact inhibition and metabolite accumulation. This technique is divided into three categories according to cell growth characteristics: centrifugal passage (suspension cells), direct pipetting passage (semi-suspension cells), and enzymatic digestion passage (adherent cells).

[0035] The term "primary culture" used in this article refers to the immediate culture of tissues or cells taken directly from an organism. At this stage, the cells retain their original basic properties, and normal cells still retain their diploid number. Cultured cells from the first to the tenth generation are collectively referred to as primary cell culture. Commonly used methods include tissue block culture and digestion culture.

[0036] In a first aspect, the present invention provides a method for in vitro construction of a recombinant human vaginal full-thickness model, comprising the following steps: S1. Human skin fibroblasts are mixed with matrix material and cultured to obtain matrix gel. The mixture is then cultured to form the lamina propria. S2. Seed VK2 / E6E7 cells onto the surface of the lamina propria; S3. Using a gas-liquid interface culture method, the VK2 / E6E7 cells seeded on the surface of the lamina propria are induced to differentiate and culture to form an epithelial layer, thus obtaining the recombinant human vaginal full-thickness model.

[0037] According to some embodiments of the present invention, prior to step S1, the in vitro construction method further includes: Pretreatment of the human skin fibroblasts (including isolation and culture): The isolated skin tissue was placed in 100-300 U / mL collagenase digestion solution and digested at 35℃-39℃ for 2-4 hours. After digestion was terminated, the cells were collected by filtration and centrifugation. The cells were washed and resuspended in culture medium and passaged to the 3rd-6th generation at 35℃-39℃ and 5% CO2.

[0038] Furthermore, the culture medium used in the pretreatment of human skin fibroblasts includes DMEM medium containing 8%-12% fetal bovine serum.

[0039] Specifically, the pretreatment of the human skin fibroblasts includes placing the separated lamina propria of skin tissue into a 200 U / mL collagenase digestion solution, digesting at 37°C for 3 hours, terminating the digestion with DMEM culture medium containing 10% fetal bovine serum (FBS), filtering through a 200-mesh sieve, centrifuging to remove the supernatant, and collecting the human skin fibroblasts; washing the human skin fibroblasts with PBS, resuspending the cell pellet in DMEM culture medium containing 10% FBS, seeding them in T75 culture flasks, and passaged at 37°C under 5% CO2 conditions for 3-6 passages, ensuring cell viability ≥90% before use; during culture, screening for morphologically uniform and actively proliferating fibroblasts, and removing senescent cells. It is understood that the pretreatment of human skin fibroblasts includes, but is not limited to, the specific pretreatment steps described above, and those skilled in the art can make appropriate adjustments according to actual circumstances and needs.

[0040] According to some embodiments of the present invention, in step S1, the method for forming the intrinsic layer includes: Human skin fibroblasts were mixed with matrix material at a volume ratio of 1:(5-15) and inoculated into a culture device. The inoculation amount of human skin fibroblasts was 2×10⁻⁶. 5 5×10 6 Each sample is placed in a culture medium at 35℃-39℃ for 1-4 hours to gel; after gelation, it is cultured in a culture medium at 35℃-39℃ and 5% CO2 for 3-5 days.

[0041] Furthermore, the culture device can be a cell culture insert, such as a Transwell chamber.

[0042] The seeding density of human skin fibroblasts in each culture device (Transwell chamber) can, for example, be 5 × 10⁶ cells / mL. 5 6×10 5 7×10 5 8×10 5 9×10 5 1×10 6 1.5 × 10 6 1, 2×10 6 One or any value within the range of any two of the above values, preferably 1×10 6 indivual.

[0043] Furthermore, the thickness of the intrinsic layer can be, for example, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, or any value within the range of any two of the above values.

[0044] Further, the culture medium used in step S1 may include DMEM medium containing 10% fetal bovine serum. Even further, the DMEM medium containing 10% fetal bovine serum is preferably a high-glucose DMEM medium (glucose content of 4500 mg / L) containing 10% fetal bovine serum.

[0045] According to some embodiments of the present invention, the human skin fibroblasts are selected from primary human skin fibroblasts or immortalized human skin fibroblasts.

[0046] According to some embodiments of the present invention, the matrix material is selected from collagen gel or Matrigel matrix gel-collagen composite matrix solution.

[0047] Furthermore, in the Matrigel matrix-collagen composite matrix solution, the volume ratio of Matrigel matrix to collagen can be 1:(3-5), preferably 1:4.

[0048] Furthermore, collagen gel can serve as a neutral collagen scaffold.

[0049] For example, the method of forming the intrinsic layer may specifically include: Human skin fibroblasts were mixed with matrix material at a volume ratio of 1:9 and seeded into Transwell chambers, resulting in a seeding density of 1 × 10⁶ human skin fibroblasts per chamber. 6 Each cell was placed in a 37°C environment and cultured for 2 hours to allow the matrix material to solidify. After gelation, the cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2 for 4 days, with the medium being changed daily. This allowed human skin fibroblasts to proliferate and spread fully in the matrix gel, forming a lamina propria with a certain mechanical strength and three-dimensional structure. The thickness of the lamina propria reached 150 μm-200 μm, and the human fibroblasts were evenly distributed in the matrix gel.

[0050] According to some embodiments of the present invention, prior to step S2, the in vitro construction method further includes: The VK2 / E6E7 cells were pretreated by adding 0.05-0.2 ng / mL recombinant human epidermal growth factor, 0.03-0.07 mg / mL bovine pituitary extract, and 0.2-0.6 mM calcium chloride to serum-free keratinocyte culture medium. The cells were cultured at 35-35℃-39℃ and 5% CO2. When the cell plating rate reached 70-90%, the cells were passaged. After passage to the 3rd-5th generation, the cells were used for model construction.

[0051] Specifically, pretreatment of the VK2 / E6E7 cells may include: using serum-free keratinocyte culture medium (SFM) supplemented with 0.1 ng / mL recombinant human epidermal growth factor (rhEGF), 0.05 mg / mL bovine pituitary extract (BPE), and 44.1 mg / L calcium chloride (CaCl2, final concentration 0.4 mM). The cells are cultured at 37°C in a 5% CO2 incubator, with the medium changed every 24 hours. When the cells reach approximately 80% plating coverage, they are passaged. After the third passage, the cells are used for model construction. It is understood that the pretreatment of VK2 / E6E7 cells includes, but is not limited to, the specific pretreatment steps described above, and those skilled in the art may make appropriate adjustments based on actual circumstances and needs.

[0052] According to some embodiments of the present invention, in step S2, VK2 / E6E7 cells are seeded on the surface of the lamina propria, and the seeding amount of VK2 / E6E7 cells is 1.25 × 10⁻⁶. 6 5×10 6 After inoculation, cells were cultured in FVU medium at 35℃-39℃ for 36-60 hours to allow for full cell adhesion.

[0053] The basal solution of the FVU medium is a mixture of DMEM medium and F12 medium at a volume ratio of (2-5):1, and the FVU medium contains: 8%-12% fetal bovine serum, 0.1-2 ng / mL epidermal growth factor, 3-15 μg / mL insulin, 5-10 μmol / mL glutamine, 20-40 μg / mL adenine, 2-8 μg / mL hydrocortisone, 50-150 μg / mL vitamin C, 10-30 ng / mL basic fibroblast growth factor, 20-40 ng / mL transferrin, 0.2-0.8 nmol / mL triiodothyronine, and 5-20 μmol / mL isoproterenol.

[0054] The volume ratio of DMEM medium to F12 medium in FVU medium can be exemplarily 2:1, 3:1, 4:1, 5:1 or any value within the range of any two of the above values, with 3:1 being the preferred volume ratio.

[0055] Furthermore, VK2 / E6E7 can be seeded onto the surface of the intrinsic layer by seeding into the upper chamber of the Transwell chamber.

[0056] The seeding amount of VK2 / E6E7 cells is 1.25 × 10⁻⁶ cells, exemplarily. 6 1, 2×10 6 1, 2.5 × 10 61, 3×10 6 1, 4×10 6 5×10 6 It can be any value within the range of any two of the above values.

[0057] Specifically, step S2 includes adjusting the cell density of pretreated VK2 / E6E7 cells with Keratinocyte-SFM medium and seeding them on the surface of the lamina propria (upper chamber of the Transwell), so that the number of cells seeded in each Transwell chamber is 2.5 × 10⁻⁶. 6 Add FVU medium and incubate at 37°C for 48 hours to allow cells to fully adhere. The FVU medium is a mixture of DMEM and F12 medium at a volume ratio of 3:1, with the following added: 10% FBS, 2 ng / mL LEGF, 10 μg / mL insulin, 8 μmol / mL glutamine, 30 μg / mL adenine, 5 μg / mL hydrocortisone, 100 μg / mL vitamin C, 20 ng / mL basic fibroblast growth factor, 30 ng / mL transferrin, 0.5 nmol / mL triiodothyronine, and 10 μmol / mL isoproterenol.

[0058] According to some embodiments of the present invention, in step S3, the induced differentiation culture includes gas-liquid interface culture for 9-12 days using FVT differentiation medium at 35℃-39℃.

[0059] Compared to FVU medium, FVT differentiation medium mainly removes epidermal growth factor and adds a certain amount of calcium chloride. The basal solution of FVT differentiation medium is a mixture of DMEM medium and F12 medium at a volume ratio of (2-5):1, and contains: 2%-8% fetal bovine serum, 5-15 μg / mL insulin, 7-10 μmol / mL glutamine, 20-40 μg / mL adenine, 3-8 μg / mL hydrocortisone, 80-120 μg / mL vitamin C, 10-30 ng / mL basic fibroblast growth factor, 25-35 ng / mL transferrin, 0.5-0.8 nmol / mL triiodothyronine, 5-15 μmol / mL isoproterenol, 1-3 μmol / mL calcium chloride, 5-100 µg / mL hyaluronic acid oligosaccharide, 2-50 µM manganese ions, and 0.1-0.5 μmol / L LTGF-β inhibitor.

[0060] In some embodiments, the volume ratio of DMEM medium to F12 medium in FVT medium can be exemplarily 2:1, 3:1, 4:1, 5:1 or any value within the range of any two of the above values, with the volume ratio preferably being 3:1.

[0061] In some embodiments, the amount of calcium chloride (CaCl2) added can be, for example, 0.5 μmol / mL, 1 μmol / mL, 1.5 μmol / mL, 2 μmol / mL, 2.5 μmol / mL, or any value within the range of any two of the above values, preferably 1.5 μmol / mL.

[0062] In some embodiments, the TGF-β inhibitor is selected from at least one of A83-01, SB-431542, and ALK5 Inhibitor II.

[0063] Specifically, in step S3, the culture medium in step S2 is replaced with differentiation medium FVT, and cultured using an air-liquid interface for 9-12 days, changing the medium daily to induce epithelial cell differentiation. The FVT differentiation medium is a mixture of DMEM and F12 medium at a volume ratio of 3:1, supplemented with 5% FBS, 10 μg / mL insulin, 8 μmol / mL glutamine, 30 μg / mL adenine, 5 μg / mL hydrocortisone, 100 μg / mL vitamin C, 20 ng / mL basic fibroblast growth factor, 30 ng / mL transferrin, 0.5 nmol / mL triiodothyronine, 10 μmol / mL isoproterenol, 50 µg / mL hyaluronic acid oligosaccharide, 25 µM manganese ions, 0.5 μmol / L A83-01, and 1.5 μmol / mL CaCl2.

[0064] According to some embodiments of the present invention, the in vitro construction method of the recombinant human vaginal full-thickness model may further include identification after culture.

[0065] Further identification included histological staining (HE staining), collagen staining (Masson staining), and glycogen staining (PAS staining) on ​​the obtained recombinant human vaginal full-thickness model. HE staining revealed the stratified squamous epithelium and lamina propria, identifying its tissue structure; Masson staining demonstrated the matrix components of the lamina propria, identifying its collagen distribution; and PAS staining demonstrated that the epithelial cells possess glycogen synthesis function, a key characteristic of the vaginal epithelium, identifying its functional features.

[0066] Secondly, the present invention provides a recombinant human vaginal full-layer model, which is prepared by the above-mentioned in vitro construction method.

[0067] According to some embodiments of the present invention, the recombinant human vaginal full-thickness model includes a lamina propria and an epithelial layer. The structure of the epithelial layer includes a basal layer, a spinous layer, and a granular layer, and a tight basement membrane connection is formed between the epithelial layer and the lamina propria.

[0068] Thirdly, the present invention provides the application of the above-described in vitro construction method or the above-described recombinant human vaginal full-layer model in the preparation of products for research on the pathogenesis of vaginal-related diseases, drug screening or toxicity assessment.

[0069] This application does not involve the diagnosis and treatment of diseases.

[0070] For example, the vaginal-related diseases include at least one of vaginitis, vaginal cancer, vaginal mucosal injury, vaginal intraepithelial neoplasia, congenital vaginal malformation, or vaginal atrophy.

[0071] For example, the pathogenesis studies include studies on the impact of pathogenic microorganisms (such as bacteria, fungi, and viruses) on the vaginal epithelial barrier function, studies on the interaction between the vaginal microecology and the host, studies on the regulation of vaginal epithelial differentiation by hormones, or studies on the mechanisms of tumor invasion and metastasis.

[0072] For example, the drug screening targets include at least one of the following: topical anti-inflammatory drugs, topical antiviral drugs, antibacterial drugs, antifungal drugs, hormonal drugs, vaginal care products, antimicrobial agents, or contraceptives.

[0073] For example, the toxicity assessment may be conducted on at least one of cosmetics, personal care products, medical devices, vaginal implant materials, or vaginal mucosal repair materials.

[0074] For example, the detection indicators for the assessment include one or more of the following: transepithelial electrical resistance, cell permeability, cell viability, lactate dehydrogenase release, expression levels of inflammatory factors (such as IL-1β, IL-6, IL-8, TNF-α), histological structural integrity, or glycogen expression levels.

[0075] Examples 1-4 of this invention at least support the protection scope of "in vitro construction method of recombinant human vaginal full-thickness model".

[0076] The “in vitro construction method of recombinant human vaginal full-thickness model” is summarized from the foregoing explanation and / or the corresponding construction methods in Examples 1-4. Therefore, those skilled in the art can reasonably presume that the “in vitro construction method of recombinant human vaginal full-thickness model”, its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it based on the existing technical level and within the scope of conventional technical means and common knowledge should all fall within the protection scope of the “in vitro construction method of recombinant human vaginal full-thickness model”.

[0077] Examples 1-4 of this invention at least support the protection scope of the "reconstructed human vaginal full-layer model".

[0078] The term "recombinant human vaginal full-layer model" is derived from the recombinant human vaginal full-layer model obtained in the foregoing explanation and / or Examples 1-4. Therefore, those skilled in the art can reasonably presume that the "recombinant human vaginal full-layer model," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of the "recombinant human vaginal full-layer model."

[0079] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides an in vitro construction method, product, and application of a recombinant human vaginal full-thickness model, which has better technical effects, specifically reflected in the following aspects: (1) High physiological relevance and tissue structure similarity: This invention uses the immortalized cell line VK2 / E6E7 derived from vaginal epithelium. Its differentiation characteristics and physiological functions are highly consistent with normal vaginal epithelial cells, and it can form a complete stratified squamous epithelial structure (basal layer, spinous layer, and granular layer), which fundamentally improves the physiological relevance of the model. At the same time, by optimizing the induction differentiation culture conditions, the epithelial cells are further induced to differentiate fully, and a tight basement membrane connection is formed between the epithelial layer and the lamina propria. The similarity of the model tissue structure to the full-thickness tissue of the human vagina is not less than 85%, which can accurately simulate the barrier function and microenvironment of the human vagina, far superior to the existing models using A431 cells.

[0080] (2) High stability and reproducibility: This invention uses human skin fibroblasts, which are readily available, easy to culture on a large scale, have strong in vitro proliferation capacity, and are stable in passage, thus reducing the problems of scarce human vaginal fibroblast sources and large batch-to-batch variations. At the same time, VK2 / E6E7 cells can be immortalized, avoiding the problems of scarce primary epithelial cell sources and limited passage, effectively improving the batch consistency and long-term stability of model preparation, with an experimental reproducibility rate of no less than 90%.

[0081] (3) The preparation process is simple, the cost is low, and it is easy to scale up: The present invention uses commercial cell lines to avoid the complex primary cell separation process. The culture conditions are mild and the operation is simple, which reduces the time and economic costs. Compared with the primary cell construction method, it greatly reduces the time and economic costs of model preparation, and can realize large-scale and standardized production to meet the needs of large-scale applications such as drug screening and disease research.

[0082] (4) Broad application prospects: The model constructed in this invention has a complete full-layer vaginal structure and stable physiological functions, and can be widely used in the study of the pathogenesis of gynecological diseases (such as vaginitis and vaginal cancer), evaluation of vaginal mucosal repair materials, screening and toxicity detection of gynecological drugs (such as topical anti-inflammatory drugs and antiviral drugs). Compared with existing models, its research results are more reliable and have greater clinical translational value. Attached Figure Description

[0083] Figure 1 This is a histological HE staining image of the recombinant human vaginal full-thickness model obtained in Example 1 of the present invention, wherein... Figure 1 Figure A and Figure 1 Figure B shows HE staining at different magnifications.

[0084] Figure 2 This is a Masson staining image of collagen expression in the recombinant human vaginal full-thickness model obtained in Example 1 of the present invention, wherein... Figure 2 Figure A and Figure 2 Figure B shows Masson staining at different magnifications.

[0085] Figure 3 This is a PAS staining image of glycogen expression in the recombinant human vaginal full-thickness model obtained in Example 1 of this invention, wherein... Figure 3 Figure A and Figure 3 Figure B shows the PAS staining at different magnifications.

[0086] Figure 4 This is a staining image of the recombinant human vaginal full-thickness model obtained in Example 2 of the present invention, wherein, Figure 4 Image A is a HE staining image. Figure 4 Image B is a Masson staining image.

[0087] Figure 5 This is a staining image of the recombinant human vaginal full-thickness model obtained in Example 3 of the present invention, wherein, Figure 5 Image A is a HE staining image. Figure 5 Image B is a Masson staining image.

[0088] Figure 6 This is a staining image of the recombinant human vaginal full-thickness model obtained in Example 4 of the present invention, wherein, Figure 6 Image A is a HE staining image. Figure 6 Image B is a Masson staining image.

[0089] Figure 7 This is a histological HE staining image of the recombinant human vaginal full-thickness model obtained in Comparative Example 1 of this invention.

[0090] Figure 8This is a histological HE staining image of the recombinant human vaginal full-thickness model obtained in Comparative Example 2 of this invention.

[0091] Figure 9 This is a histological HE staining image of the recombinant human vaginal full-thickness model obtained in Comparative Example 3 of this invention.

[0092] Figure 10 This is a histological HE staining image of the recombinant human vaginal full-thickness model obtained in Comparative Example 4 of this invention.

[0093] Figure 11 This is a histological HE staining image of the recombinant human vaginal full-thickness model obtained in Comparative Example 5 of this invention. Detailed Implementation

[0094] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0095] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0096] Data analysis and statistical analysis were performed using professional data processing software, and significance analysis was conducted using one-way ANOVA. P <0.05 indicates a significant difference.

[0097] The main reagents used in the following examples are shown in Table 1.

[0098] Table 1

[0099] Example 1 Pretreatment of primary human skin fibroblasts: Isolated skin tissue (from healthy individuals with signed informed consent) was placed in 200 U / mL collagenase digestion solution and digested at 37°C for 3 hours. Digestion was terminated using DMEM culture medium containing 10% fetal bovine serum (FBS). The cells were filtered through a 200-mesh sieve, centrifuged to remove the supernatant, and the primary human skin fibroblasts were collected. The primary human skin fibroblasts were washed with PBS, and the cell pellet was resuspended in DMEM culture medium containing 10% FBS. The cells were then seeded in T75 culture flasks and cultured at 37°C under 5% CO2 conditions. After passage 4, the cells were kept viable at ≥90% for use. During the culture process, morphologically uniform and actively proliferating fibroblasts were selected, and senescent cells were removed.

[0100] VK2 / E6E7 cells were pretreated using serum-free keratinocyte culture medium (SFM) supplemented with 0.1 ng / mL recombinant human epidermal growth factor (rhEGF), 0.05 mg / mL bovine pituitary extract (BPE), and 44.1 mg / L calcium chloride (CaCl2, final concentration 0.4 mM). Cells were incubated at 37°C in a 5% CO2 incubator, with the medium changed every 24 hours. Cells were passaged when the plating rate reached approximately 80%, and then cultured to the third generation for further use.

[0101] Step S1: Adjust the density of the pretreated primary human skin fibroblasts cell suspension using DMEM containing 10% fetal bovine serum. Mix the primary human skin fibroblasts with a neutral collagen scaffold at a volume ratio of 1:9 and seed them into Transwell chambers, ensuring that the seeding density of human skin fibroblasts in each chamber is 1 × 10⁶ cells / mL. 6 Each cell was placed at 37°C and cultured for 2 hours to allow the neutral collagen scaffold of the matrix material to solidify. After gelation, the cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2 for 4 days, with the medium being changed daily. This allowed human skin fibroblasts to proliferate and spread fully in the matrix gel, forming a lamina propria with a certain mechanical strength and three-dimensional structure. The thickness of the lamina propria reached 150 μm-200 μm, and the human fibroblasts were evenly distributed in the matrix gel.

[0102] Step S2: Adjust the cell density of the pretreated VK2 / E6E7 cells using Keratinocyte-SFM medium and seed them onto the surface of the lamina propria (upper chamber of the Transwell), ensuring that each Transwell chamber contains 2.5 × 10⁶ cells. 6Add FVU medium and incubate at 37°C for 48 hours to allow cells to fully adhere. The FVU medium is a mixture of DMEM and F12 medium at a volume ratio of 3:1, with the following added: 10% FBS, 2 ng / mL EGF, 10 μg / mL insulin, 8 μmol / mL glutamine, 30 μg / mL adenine, 5 μg / mL hydrocortisone, 100 μg / mL vitamin C, 20 ng / mL basic fibroblast growth factor, 30 ng / mL transferrin, 0.5 nmol / mL triiodothyronine, and 10 μmol / mL isoproterenol.

[0103] Step S3: Replace the culture medium in step S2 with differentiation medium FVT and use an air-liquid interface culture method to induce differentiation of the VK2 / E6E7 cells seeded on the surface of the lamina propria for 9 days. During this period, the culture medium is changed once a day to allow the VK2 / E6E7 cells to differentiate into a stratified squamous epithelial structure and form an epithelial layer, thereby obtaining the recombinant human vaginal full-thickness model containing the lamina propria and the epithelial layer. The FVT differentiation medium consisted of a mixture of DMEM and F12 medium at a volume ratio of 3:1, supplemented with 5% FBS, 10 μg / mL insulin, 8 μmol / mL glutamine, 30 μg / mL adenine, 5 μg / mL hydrocortisone, 100 μg / mL vitamin C, 20 ng / mL basic fibroblast growth factor, 30 ng / mL transferrin, 0.5 nmol / mL triiodothyronine, 10 μmol / mL isoproterenol, 50 µg / mL hyaluronic acid oligosaccharide, 25 µM manganese ions, 0.5 μmol / L A83-01, and 1.5 μmol / mL CaCl2.

[0104] Example 2 The only difference from Example 1 is that immortalized human skin fibroblasts were used instead of primary human skin fibroblasts. The immortalized human skin fibroblasts underwent pretreatment: the frozen immortalized human skin fibroblasts were thawed, centrifuged, and resuspended in a 37°C water bath. All resuspended cells were added to a culture flask and gently shaken to ensure even distribution. The flask was then placed in a 37°C, 5% CO2 incubator. After overnight adhesion, the medium was changed. The cells were passaged in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for three passages, ensuring cell viability ≥90% before use. During culture, morphologically uniform and actively proliferating fibroblasts were selected, and senescent cells were removed. The remaining in vitro construction methods were essentially the same as in Example 1.

[0105] Example 3 The only difference from Example 1 is that in step S2, the FVU medium is a mixture of DMEM and F12 medium at a volume ratio of 3:1, with the addition of 8% fetal bovine serum, 0.1 ng / mL epidermal growth factor, 3 μg / mL insulin, 5 μmol / mL glutamine, 20 μg / mL adenine, 2 μg / mL hydrocortisone, 50 μg / mL vitamin C, 10 ng / mL basic fibroblast growth factor, 20 ng / mL transferrin, 0.2 nmol / mL triiodothyronine, and 5 μmol / mL isoproterenol; in step S3, the FVT differentiation medium is a mixture of DMEM and F12 medium at a volume ratio of 3:1, with the addition of 2% FBS, 5 μg / mL insulin, 7 μmol / mL glutamine, 20 μg / mL adenine, 3 μg / mL hydrocortisone, 80 μg / mL vitamin C, and 10 ng / mL basic fibroblast growth factor, 20 ng / mL transferrin, 0.2 nmol / mL triiodothyronine, and 5 μmol / mL isoproterenol. The following ingredients were used in the in vitro construction: 25 ng / mL basic fibroblast growth factor, 0.5 nmol / mL triiodothyronine, 5 μmol / mL isoproterenol, 5 µg / mL hyaluronic acid oligosaccharide, 2 µM manganese ions, 0.1 μmol / L A83-01, and 1 μmol / mL CaCl2. The remaining in vitro construction methods were essentially the same as in Example 1.

[0106] Example 4 The only difference from Example 1 is that in step S2, the FVU medium is a mixture of DMEM and F12 medium at a volume ratio of 3:1, with the addition of 12% fetal bovine serum, 2 ng / mL epidermal growth factor, 15 μg / mL insulin, 10 μmol / mL glutamine, 40 μg / mL adenine, 8 μg / mL hydrocortisone, 150 μg / mL vitamin C, 30 ng / mL basic fibroblast growth factor, 40 ng / mL transferrin, 0.8 nmol / mL triiodothyronine, and 20 μmol / mL isoproterenol; in step S3, the FVT differentiation medium is a mixture of DMEM and F12 medium at a volume ratio of 3:1, with the addition of 8% fetal bovine serum, 15 μg / mL insulin, 10 μmol / mL glutamine, 40 μg / mL adenine, 8 μg / mL hydrocortisone, 120 μg / mL vitamin C, 30 ng / mL basic fibroblast growth factor, 40 ng / mL transferrin, 0.8 nmol / mL triiodothyronine, and 20 μmol / mL isoproterenol. The following ingredients were used in the in vitro construction: 1 ng / mL basic fibroblast growth factor, 35 ng / mL transferrin, 0.8 nmol / mL triiodothyronine, 15 μmol / mL isoproterenol, 100 µg / mL hyaluronic acid oligosaccharide, 50 µM manganese ions, 0.5 μmol / L A83-01, and 3 μmol / mL calcium chloride. The remaining in vitro construction methods were essentially the same as in Example 1.

[0107] Comparative Example 1 Model construction based on existing technology (CN113293126A) (positive control): A vaginal mucosal model was constructed according to the method in Example 4 of patent CN113293126A. The epithelial layer was constructed using the vulvar epidermoid carcinoma cell line A431, and the stromal layer was constructed using primary human vaginal fibroblasts.

[0108] Comparative Example 2 The only difference from Example 1 is that CaCl2 is not added to the FVT differentiation medium in step S3. The rest of the in vitro construction methods are basically the same as in Example 1.

[0109] Comparative Example 3 The only difference from Example 1 is that hyaluronic acid oligosaccharides are not added to the FVT differentiation medium in step S3. The rest of the in vitro construction methods are basically the same as in Example 1.

[0110] Comparative Example 4 The only difference from Example 1 is that manganese ions are not added to the FVT differentiation medium in step S3. The rest of the in vitro construction methods are basically the same as in Example 1.

[0111] Comparative Example 5 The only difference from Example 1 is that A83-01 is not added to the FVT differentiation medium in step S3. The rest of the in vitro construction methods are basically the same as in Example 1.

[0112] The models obtained by the in vitro construction methods in Examples 1-4 and Comparative Examples 1-5 were subjected to the following tests.

[0113] Example 1: Model Identification (1) HE staining: Observe the tissue structure of the model to confirm whether a complete stratified squamous epithelium (including basal layer, spinous layer, granular layer) and lamina propria structure has been formed.

[0114] The full-thickness vaginal model was fixed in 4% paraformaldehyde solution, followed by paraffin embedding and section staining for histological examination. ① Paraffin embedding and sectioning: After fixing the tissue for more than 24 hours, the tissue was sequentially immersed in 75%, 85%, and 95% anhydrous ethanol for 60 minutes each, then sequentially immersed in two 100% anhydrous ethanol solutions for dehydration. After clearing, the tissue was sequentially immersed in xylene I and xylene II for 6 minutes each. After clearing, the tissue block was sequentially immersed in: soft wax I (30 minutes), soft wax II (30 minutes), and hard wax (1 hour). After paraffin embedding, the molten wax was poured into the embedding cassette, and the tissue block was quickly picked up with forceps, cut side down, and placed in the center of the cassette. It was removed after the paraffin had completely solidified and hardened. Ten sections were prepared from each specimen, with a section thickness of approximately 5 μm. The sections were placed on glass slides and baked in a slide oven at 60°C for 60 minutes. Before staining, the sections were placed in a 37°C oven overnight or baked at 60°C for 2 hours. ② H&E staining of tissue sections: Immerse the sections sequentially in xylene I (10 min); xylene II (10 min); anhydrous ethanol I (5 min); anhydrous ethanol II (5 min); 95% ethanol (5 min); 90% ethanol (5 min); 80% ethanol (5 min); 70% ethanol (5 min); wash with distilled water. Stain the sections with hematoxylin for 3-8 min, then rinse with tap water. Stain the sections with eosin for 1-3 min, then rinse with tap water. Dehydrate and clear the sections sequentially in 95% ethanol I (5 min); 95% ethanol II (5 min); anhydrous ethanol I (5 min); anhydrous ethanol II (5 min); xylene I (5 min); xylene II (5 min). Remove the sections from the xylene and allow them to air dry slightly. Mount with neutral resin.

[0115] (2) Masson staining: Observe the distribution and density of collagen fibers in the lamina propria.

[0116] The sections were sequentially immersed in xylene I (10 min); xylene II (10 min); anhydrous ethanol I (5 min); anhydrous ethanol II (5 min); 95% ethanol (5 min); 90% ethanol (5 min); 80% ethanol (5 min); 70% ethanol (5 min); and washed with distilled water. The sections were then stained with hematoxylin for 3-8 min and rinsed with tap water. Differentiation was performed with 1% hydrochloric acid ethanol for 1-2 seconds, followed by blueing with tap water for 3-5 min, and then rinsed with distilled water. The sections were then stained with Ponceau S and Acidic Fuchsin for 5-10 min, quickly washed twice with distilled water, differentiated with phosphomolybdic acid-phosphotungstic acid solution for 5-10 min, immersed in aniline blue (5-10 min), and treated with 0.2%-1% glacial acetic acid for 1 min. The sections were sequentially immersed in 95% ethanol I for 5 min; 95% ethanol II for 5 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; xylene I for 5 min; and xylene II for 5 min to dehydrate and clear the sections. The sections were then removed from the xylene and allowed to dry slightly before being mounted with neutral resin.

[0117] (3) PAS staining: Observe the expression of glycogen in the epithelial layer and assess the physiological function of the model.

[0118] a. Take model sections and dewax them to water using standard methods; b. Oxidize with 3% periodic acid solution for 10 minutes, then rinse with tap water; c. Add Schiff's reagent and stain at room temperature in the dark for 15 minutes, then rinse with tap water; d. Counterstain cell nuclei with hematoxylin solution for 5 minutes, rinse with tap water, differentiate with hydrochloric acid ethanol for 3 seconds, then rinse again with tap water to regain blue color; e. Dehydrate in a gradient (70%, 80%, 90%, 100% ethanol), clear (xylene), and mount with neutral resin; f. Observe under an optical microscope.

[0119] Results analysis: The staining results of the recombinant human vaginal full-thickness model in Example 1 are shown in the figure. Figures 1 to 3 HE staining showed that the model's epithelial structure was clear, containing a complete basal layer, spinous layer, and granular layer, with orderly arranged epithelial cells and tight junctions between the epithelial layer and the lamina propria. Masson staining showed abundant and evenly distributed collagen fibers in the lamina propria. PAS staining showed positive purplish-red glycogen granules in the cytoplasm of the epithelial layer, simulating the metabolic characteristics of the human vaginal epithelium. These results indicate that Example 1 successfully constructed a recombinant human vaginal full-thickness model that highly simulates the human vagina. The staining results of Examples 2-4 are shown below. Figures 4 to 6 In embodiments 2-4 of the present invention, a complete stratified squamous epithelium (with the basal layer, spinous layer, and granular layer clearly visible) is formed and a tight connection is formed between the epithelial layer and the lamina propria.

[0120] The HE staining results of comparative examples 1-5 are shown below. Figures 7 to 11 Among them, comparative example 1 ( Figure 7 In contrast example 2, the epithelial layer was not fully differentiated, and some parts infiltrated into the lamina propria. Figure 8 In contrast, the epithelial layer in the sample was not fully differentiated, the cell connections were loose, and the epidermal layer was missing in some areas. (Comparative example 3) Figure 9 In Comparative Example 4, separation of the epithelial layer and lamina propria was observed, with visible local gaps and discontinuous basement membrane. Figure 10 The surface of the mesothelial layer is slightly rough, and separation of the epithelial layer and lamina propria is observed, with local gaps visible. The basement membrane is discontinuous. (Comparative Example 5) Figure 11 The epithelial layer is not fully differentiated and the thickness of the epithelial layer is uneven.

[0121] The above results demonstrate that, compared to comparative examples 1-5, the recombinant human vaginal full-thickness model constructed in this invention has a higher similarity to human vaginal full-thickness tissue. Furthermore, the FVT differentiation culture medium provided by this invention exhibits a good synergistic effect among Ca ions, hyaluronic acid oligosaccharides, manganese ions, and TGF-β inhibitors, which can systematically promote full differentiation of the epithelial layer, enhance barrier function, and stabilize the tight junction of the epithelial-lamina propria interface.

[0122] Evaluation of model stability and batch consistency in Example 2 To verify the stability and reproducibility of the model constructed in this invention, the model was constructed in three independent batches (Batch1, Batch2, Batch3) according to the methods of Example 1 and Comparative Example 1, with 6 parallel samples in each batch. The following key indicators were tested and statistically analyzed.

[0123] Key metrics include: (1) Transepithelial resistance (TEER value): The TEER value of the model was detected using an epithelial resistance meter (Millicell ERS-2) to assess the integrity of epithelial barrier function.

[0124] (2) Epithelial thickness: The model was stained with HE, and five fields of view were randomly selected under an optical microscope (×200x) to measure the epithelial thickness using image analysis software. The average value was taken.

[0125] (3)Laminar thickness: The thickness of the lamina propria was measured in HE-stained sections and the average value was taken.

[0126] The test results are shown in Table 2.

[0127] Table 2

[0128] The above results show that, compared with Comparative Example 1, the inter-batch coefficient of variation (CV%) of the TEER values ​​of each batch model in Example 1 is ≤1.055%, and the inter-batch CV% of the epithelial layer thickness and the intrinsic layer thickness is ≤1.2%, indicating that the model constructed by the present invention has excellent batch consistency and stability.

[0129] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for in vitro construction of a recombinant human vaginal full-thickness model, characterized in that, Includes the following steps: S1. Human skin fibroblasts are mixed with matrix material and cultured to obtain matrix gel. The mixture is then cultured to form the lamina propria. S2. Seed VK2 / E6E7 cells onto the surface of the lamina propria; S3. Using a gas-liquid interface culture method, the VK2 / E6E7 cells seeded on the surface of the lamina propria are induced to differentiate and culture to form an epithelial layer, thus obtaining the recombinant human vaginal full-thickness model.

2. The in vitro construction method according to claim 1, characterized in that, In step S1, the method for forming the intrinsic layer includes: Human skin fibroblasts were mixed with matrix material at a volume ratio of 1:(5-15) and seeded into a culture device. The seeding amount of human skin fibroblasts was 5 × 10⁻⁶. 5 2×10 6 Each sample is placed in a culture medium at 35℃-39℃ for 1-4 hours to gel; after gelation, it is cultured in a culture medium at 35℃-39℃ and 5% CO2 for 3-5 days.

3. The in vitro construction method according to claim 1, characterized in that, In step S2, the seeding density of VK2 / E6E7 cells was 1.25 × 10⁻⁶. 6 5×10 6 Individuals were inoculated and cultured in FVU medium at 35℃-39℃ for 36-60 hours after inoculation.

4. The in vitro construction method according to claim 1, characterized in that, The basal solution of the FVU medium is a mixture of DMEM medium and F12 medium at a volume ratio of (2-5):1, and contains: 8%-12% fetal bovine serum, 0.1-2 ng / mL epidermal growth factor, 3-15 μg / mL insulin, 5-10 μmol / mL glutamine, 20-40 μg / mL adenine, 2-8 μg / mL hydrocortisone, 50-150 μg / mL vitamin C, 10-30 ng / mL basic fibroblast growth factor, 20-40 ng / mL transferrin, 0.2-0.8 nmol / mL triiodothyronine, and 5-20 μmol / mL isoproterenol.

5. The in vitro construction method according to claim 1, characterized in that, In step S3, the induced differentiation culture includes gas-liquid interface culture at 35℃-39℃ for 9-12 days using FVT differentiation medium.

6. The in vitro construction method according to claim 5, characterized in that, The basal solution of the FVT differentiation medium is a mixture of DMEM medium and F12 medium at a volume ratio of (2-5):1, and contains: 2%-8% fetal bovine serum, 5-15 μg / mL insulin, 7-10 μmol / mL glutamine, 20-40 μg / mL adenine, 3-8 μg / mL hydrocortisone, 80-120 μg / mL vitamin C, 10-30 ng / mL basic fibroblast growth factor, 25-35 ng / mL transferrin, 0.5-0.8 nmol / mL triiodothyronine, 5-15 μmol / mL isoproterenol, 1-3 μmol / mL calcium chloride, 5-100 µg / mL hyaluronic acid oligosaccharide, 2-50 µM manganese ions and 0.1-0.5 μmol / L TGF-β inhibitor.

7. The in vitro construction method according to claim 1, characterized in that, Prior to step S1, the in vitro construction method further includes: Pretreatment of the human skin fibroblasts: The isolated skin tissue was placed in 100-300 U / mL collagenase digestion solution and digested at 35℃-39℃ for 2-4 hours. After digestion was terminated, the cells were collected by filtration and centrifugation. The cells were washed and resuspended in DMEM medium containing 8%-12% fetal bovine serum and passaged at 35℃-39℃ and 5% CO2 to the 3rd-6th generation.

8. The in vitro construction method according to claim 1, characterized in that, Prior to step S2, the in vitro construction method further includes: The VK2 / E6E7 cells were pretreated by adding 0.05-0.2 ng / mL recombinant human epidermal growth factor, 0.03-0.07 mg / mL bovine pituitary extract, and 0.2-0.6 mM calcium chloride to serum-free keratinocyte culture medium. The cells were cultured at 35℃-39℃ and 5% CO2. When the cell plating rate reached 70-90%, the cells were passaged. After passaged to the 3rd-5th generation, the cells were used for model construction.

9. The in vitro construction method according to claim 1, characterized in that, The human skin fibroblasts are selected from primary human skin fibroblasts or immortalized human skin fibroblasts.

10. The in vitro construction method according to claim 1, characterized in that, The matrix material is selected from collagen gel or Matrigel matrix gel-collagen composite matrix solution.

11. A reconstructed full-thickness model of the human vagina, characterized in that, The recombinant human vaginal full-thickness model is obtained by the in vitro construction method according to any one of claims 1-10.

12. The use of the in vitro construction method according to any one of claims 1-10 or the recombinant human vaginal full-thickness model according to claim 11 in the preparation of products for research on the pathogenesis of vaginal-related diseases, drug screening or toxicity assessment.

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