3D lip model, construction method and lip product irritancy test method

Abstract

The application belongs to the technical field of tissue engineering and skin irritancy detection, and more particularly relates to a 3D lip-like epithelial model, a construction method and a lip product irritancy test method. The application obtains a 3D lip-like epithelial model with a non-keratinized stratified squamous epithelial structure by pre-treating a cell culture chamber, inoculating epidermal primary keratinocytes, and sequentially inducing cell stratification and differentiation through immersion culture and gas-liquid interface culture. The model is composed of a basal layer, a spinous layer and a non-keratinized squamous epithelial layer and has no typical granular layer, and is highly consistent with human lip tissue. The sensitivity of the model in detecting irritant components is significantly better than that of a conventional reconstructed epidermis model, and the model is suitable for irritancy detection of various types of lip samples. The lip product irritancy test method based on the model fills the industry gap in the exclusive detection method of lip product irritancy, and provides a scientific and reliable technical path for the safety detection of lip products and related compounds.

Description

Technical Field

[0001] This application belongs to the fields of tissue engineering and skin irritation testing technology, and more specifically, relates to a 3D lip-like epithelial model, its construction method, and a method for testing the irritation of lip products. Background Technology

[0002] The lips are one of the most frequently contacted areas by cosmetics and personal care products (such as lipsticks, lip glosses, lip balms, lip serums, and lip makeup removers), and their safety is directly related to consumer health. Unlike regular skin, the lips have a unique physiological structure. Lip tissue is composed of non-keratinized stratified squamous epithelium, lacking a typical granular layer structure, making its barrier function relatively fragile. This special tissue structure makes the lips significantly more sensitive to exogenous irritants than regular skin. Therefore, lip products require specific irritation assessments based on their physiological characteristics before being marketed to ensure the product's safety for the lip skin.

[0003] Currently, various in vitro epidermal models have been developed for skin irritation testing. For example, patent applications CN107164315A and CN120608011A disclose reconstructed epidermal models, and patent application CN120005807A discloses full-thickness skin models. (The literature "Application of an in vitro reconstructed human skin on cosmetics in skin irritation tests") Journal of Cosmetic Dermatology The study reported a method for assessing cosmetic irritation using the EpiSkin® model combined with the MTT assay in 2021 (6). However, these models are all based on conventional skin (trunk / limbs), and there is currently a lack of a dedicated epithelial model for the lip structure. Therefore, using the above-mentioned conventional epithelial models to test the irritation of lip products in practice has the following drawbacks: (1) Mismatch in model structure: Conventional reconstructed epidermal models have a complete epidermal layer structure, which presents the basal layer, spinous layer, granular layer and well-developed and dense stratum corneum from the inside out; while the natural tissue of the lip has no granular layer and the stratum corneum is extremely thin. The two structures are significantly different and cannot truly reflect the actual response of the lip skin to the product.

[0004] (2) Insufficient sensitivity: Conventional recombinant epidermal models have limited ability to identify low concentrations of irritating ingredients, and can usually only detect SDS / SLS concentrations of 2% or higher, which can easily lead to "false negatives" in the irritation assessment results of lip products.

[0005] (3) Lack of a dedicated testing program for lips: Existing cosmetic irritation testing programs are mainly designed for general skin care products and have not established a dedicated testing system for lip products, which cannot meet the testing needs of lip products.

[0006] Therefore, there is a need to provide a 3D lip-like epithelial model whose tissue structure closely matches that of the human lip, to establish an irritation testing method based on this model, and to formulate lip-specific testing parameters and judgment criteria to address the industry pain point of distorted irritation detection of lip products and related compounds. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this application aims to provide a 3D lip-like epithelial model, a construction method, and a method for testing the irritation of lip products. This aims to solve problems in existing technologies, such as distorted irritation assessment results due to the lack of a lip model, insufficient sensitivity for detecting low-concentration irritating ingredients, and the lack of a lip-specific testing system.

[0008] To achieve the above objectives, in a first aspect, this application provides a method for constructing a 3D lip-like epithelial model, comprising the following steps: S1. The cell culture chambers are sequentially subjected to hydrophilic treatment, sterile water incubation, and collagen coating treatment to obtain pretreated cell culture chambers; S2. Primary epidermal keratinocytes are seeded into the pretreated cell culture chamber and immersed in the culture chamber to allow the cells to proliferate on the filter membrane of the cell culture chamber and form a confluent monolayer of cells. S3. The cell culture chamber loaded with the above monolayer cells is subjected to gas-liquid interface culture to allow the monolayer cells to differentiate into a 3D model with a non-keratinized stratified squamous epithelial structure, thus obtaining the above 3D lip-like epithelial model.

[0009] Preferably, in step S2, the primary epidermal keratinocytes are seeded in the pretreated cell culture chamber in the form of a cell suspension, and the concentration of the cell suspension is 1×10⁻⁶. 6 ~2×10 6 per mL.

[0010] Preferably, in step S2, the immersion culture medium used in the above immersion culture includes 154 basal medium and also includes trisodium 2-phosphate-L-ascorbic acid.

[0011] Preferably, in step S2, during the above-mentioned immersion culture process, the immersion culture medium is replaced with fresh medium every 2 to 3 days.

[0012] Preferably, in step S2, the immersion culture time is 3 to 5 days.

[0013] Preferably, in step S3, the conditions for the gas-liquid interface culture are as follows: epidermal differentiation culture medium is added to the lower chamber of the cell culture chamber, and no culture medium is added to the upper chamber of the cell culture chamber.

[0014] Preferably, in step S3, during the above-mentioned gas-liquid interface culture process, the above-mentioned epidermal differentiation culture medium is replaced with fresh medium every 1 to 2 days.

[0015] Preferably, in step S3, the incubation time for the gas-liquid interface is 3 to 7 days.

[0016] Preferably, the above-mentioned epidermal differentiation culture medium includes a basal culture medium and further comprises one or more of the following: growth factor supplements, antioxidants, lipid supplements, β-adrenergic receptor agonists, amino acid supplements, and carrier proteins. The aforementioned basal culture medium comprises a mixture of high-glucose DMEM medium and 154 basal culture medium; The above-mentioned growth factor supplements include HKGS; The aforementioned antioxidants include trisodium 2-phosphate-L-ascorbic acid; The above-mentioned lipid supplements include one or more of palmitic acid, oleic acid, linoleic acid, arachidonic acid, and dl-α-tocopherol acetate; The aforementioned β-adrenergic receptor agonists include isoproterenol hydrochloride; The above-mentioned amino acid supplements contain L-serine and / or L-carnitine; The aforementioned carrier protein includes bovine serum albumin.

[0017] More preferably, the above-mentioned epidermal differentiation culture medium comprises the following components: A basal medium with a volume ratio of 1:1 between high-glucose DMEM medium and 154 basal medium; HKGS with a volume percentage of 0.5% to 2%; Trisodium 2-phosphate-L-ascorbic acid at a concentration of 45~60 μg / mL; Isoproterenol hydrochloride at a concentration of 5-15 μM; L-serine at a concentration of 8-12 mM; L-carnitine at a concentration of 8-12 μM; Fatty acid-free bovine serum albumin with a mass-volume percentage of 0.05%~0.2%; Palmitic acid at a concentration of 20-30 μM; Oleic acid at a concentration of 20~30μM; Linoleic acid at a concentration of 10~20μM; Arachidonic acid at a concentration of 5-10 μM; and dl-α-tocopherol acetate at a concentration of 20-25 μM.

[0018] Secondly, this application provides a 3D lip-like epithelial model, which is obtained by the above-described construction method.

[0019] Thirdly, this application provides a lip product irritation test kit, including the aforementioned 3D lip-like epithelial model.

[0020] Fourthly, this application provides a method for testing the irritation of lip products based on a 3D lip-like epithelial model, comprising the following steps: (1) The test sample was applied to the surface of the above-mentioned 3D lip-like epithelial model and incubated for culture, and a negative control group and a positive control group were set up; (2) After incubation, the above 3D lip-like epithelial model was cleaned, cell viability was detected, and the relative cell viability of the test group relative to the negative control group was calculated; (3) Verify the validity of the data of the negative control group and the positive control group. If the verification is successful, determine the irritation of the lip product based on the relative cell viability of the test group to obtain the irritation type.

[0021] Preferably, the incubation time in step (1) is 18-24 hours; Preferably, the method for detecting cell viability in step (2) includes the MTT assay, XTT assay, or ATP assay; Preferably, the method for determining the lip irritation in step (3) is as follows: if the relative cell viability of the test product is ≤50%, it is determined to be lip irritating; if the relative cell viability of the test product is >50%, it is determined to be non-irritating.

[0022] Preferably, the above-mentioned lip product irritation testing method further includes an inflammation risk assessment step: After the incubation culture was completed, the expression level of IL-1α in the culture medium used for incubation culture was detected; When the relative cell viability of the above-mentioned test product is >50% and the IL-1α expression level is significantly higher than that of the negative control group, it suggests that the above-mentioned test product has a potential risk of inflammatory response.

[0023] Fifthly, this application provides an application of the above-mentioned 3D lip-like epithelial model in the lip safety testing or efficacy testing of pharmaceuticals or cosmetics.

[0024] In summary, the technical solutions conceived in this application have the following main technical advantages compared with the prior art: (1) This application successfully constructed a 3D lip-like epithelial model with a non-keratinized stratified squamous epithelial structure by subjecting cell culture chambers to hydrophilic treatment, sterile water incubation, and collagen coating treatment, and then seeding primary epidermal keratinocytes into the pretreated cell culture chambers for immersion culture and gas-liquid interface culture. The model consists of a basal layer, a spinous layer, and a non-keratinized squamous epithelial layer without a typical granular layer, and its histological characteristics are highly consistent with the natural tissue of the human lip. Compared with conventional reconstructed epidermal models, the model of this application exhibits significantly different phenotypic characteristics in terms of tissue structure and degree of keratinization, providing a more human-matched model tool for in vitro research and product testing related to lip tissue.

[0025] (2) The 3D lip-like epithelial model constructed in this application exhibits excellent sensitivity in the detection of irritation in lip products. It can effectively identify low-concentration irritant components that cannot be detected by conventional body epithelial models, avoiding false negatives that may occur in the irritation assessment of lip products and solving the problem of distortion in the irritation assessment of lip products. At the same time, the model has good applicability to various types of samples, covering positive control samples, negative control samples, and various lip products such as lip gloss and makeup remover gel, providing a more accurate detection tool for the irritation assessment of lip products and related compounds.

[0026] (3) The histological characteristics of the 3D lip-like epithelial model constructed in this application are highly consistent with the natural tissue of the human lip, and can more realistically reflect the effect of the analyte on the lip tissue. The advantages of this 3D lip-like epithelial model in terms of structural biomimicry and functional stability make it a potential application element in biosensors. Based on this, this application provides a reliable cell model basis for the subsequent development of biosensors for rapid detection and real-time monitoring of lip skin irritation, and also opens up a new technical path for the further integration and automation of in vitro detection technology.

[0027] (4) The lip irritation testing method based on a 3D lip-like epithelial model established in this application enables a specialized assessment of the lip irritation of the test substance, filling the industry gap in specialized detection methods for lip irritation. It avoids the judgment bias caused by directly applying general parameters, significantly improves the accuracy and reliability of the test results, and enhances the correlation between the lip irritation result determination and the actual safety of the product. At the same time, this testing method has good compatibility with single compounds and various lip products, and supports the selection and combination of multiple detection indicators, providing flexible expansion space. It can meet the irritation assessment needs of different categories of lip-related samples, providing a scientific and reliable technical path for the safety testing of lip products and related compounds. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating the method for constructing the 3D lip-like epithelial model provided in this application; Figure 2 This is an image showing the HE staining results of the 3D lip-like epithelial model prepared according to the embodiments of this application; Figure 3 This is an HE staining result of the 3D lip-like epithelial model prepared in Comparative Example 1 of this application; Figure 4 This is an HE staining result of the 3D lip-like epithelial model prepared in Comparative Example 2 of this application; Figure 5 This is a bar chart showing the relative cell viability of different types of lip test samples on a 3D lip-like epithelial model determined by the MTT assay. Figure 6 This represents the expression level of IL-1α in the culture medium after incubation with the sensory stimulant test groups (capsaicin and menthol), where * represents... p <0.05. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0030] In the description of this application, it should be understood that the term "and / or" describes a relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. The symbol " / " in this document indicates that the related objects are in an "or" relationship; for example, A / B means A or B.

[0031] In the description of the embodiments in this application, the words "exemplary" or "for example" are used to indicate that they are examples, illustrations, or descriptions. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design options. Specifically, the use of the words "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0032] In the description of the embodiments in this application, unless otherwise stated, "multiple" means two or more.

[0033] In this application, "significantly higher than" means that the difference is statistically significant compared with the negative control group. p <0.05). The above statistical analysis was performed using an independent samples t-test.

[0034] like Figure 1As shown, this application provides a method for constructing a 3D lip-like epithelial model, including the following steps: S1. The cell culture chambers are sequentially subjected to hydrophilic treatment, sterile water incubation, and collagen coating treatment to obtain pretreated cell culture chambers; S2. Primary epidermal keratinocytes are seeded into the pretreated cell culture chamber and immersed in the culture chamber to allow the cells to proliferate on the filter membrane of the cell culture chamber and form a confluent monolayer of cells. S3. The cell culture chamber loaded with the above-mentioned monolayer cells is subjected to gas-liquid interface culture, so that the monolayer cells differentiate into a 3D model with a non-keratinized stratified squamous epithelial structure, thus obtaining the above-mentioned 3D lip-like epithelial model.

[0035] In this application, the aforementioned cell culture chamber is an insertable cell culture plug-in (e.g., Transwell series chambers or similar functional culture plug-ins), which is cup-shaped and can be placed in the wells of a multi-well culture plate (e.g., a 6-well, 12-well, or 24-well plate). The bottom of the cell culture chamber is equipped with a porous semi-permeable membrane (also referred to as a filter membrane or support membrane). This porous membrane serves as a supporting interface for cell adhesion and growth, while allowing free exchange of nutrients, metabolites, and soluble factors between the upper and lower chambers. In some embodiments, the porous semi-permeable membrane is made of polyester (PET) or polycarbonate (PC). Those skilled in the art can select an appropriate membrane pore size based on the adhesion characteristics of the target cells and the experimental purpose (e.g., co-culture, migration / invasion experiments, or three-dimensional model construction).

[0036] In some embodiments, the cell culture chambers described above undergo pretreatment steps such as hydrophilic treatment, sterile water incubation, and collagen coating before use to improve the wettability of the porous membrane surface and cell adhesion properties. The pretreated cell culture chambers have a porous membrane surface at the bottom that is more conducive to the uniform adhesion, spreading, and subsequent stratified differentiation of primary epidermal keratinocytes.

[0037] In some embodiments, step S1 includes immersing the cell culture chamber in an alcohol solution. This hydrophilic treatment refers to altering the surface properties of the filter membrane at the bottom of the cell culture chamber using physical or chemical methods, transforming the originally hydrophobic membrane into a hydrophilic one. Simultaneously, it removes residual air from the filter membrane, ensuring that subsequently inoculated cells can spread evenly and adhere firmly to the membrane surface. In some embodiments, the alcohol solution is selected from ethanol, isopropanol, or combinations thereof, with a volume fraction of 40%–60%, and the immersion time is 1–5 hours. It is understood that, considering the permeation rate of the alcohol solution and the thickness and porosity of the filter membrane, those skilled in the art can adaptively adjust the volume fraction of the alcohol solution and the immersion time, all of which fall within the scope of this application.

[0038] In some embodiments, the sterile water incubation time in step S1 is 8-12 hours. The filter membrane treated with alcohol solution may retain residual alcohol solution; if not completely removed, this can be toxic to subsequently inoculated cells, affecting cell adhesion and growth. Sterile water incubation utilizes diffusion to fully replace and remove the residual alcohol solution, while also maintaining the filter membrane in a wetted state. It is understood that those skilled in the art can appropriately adjust the sterile water incubation time based on factors such as the filter membrane thickness, porosity, and initial concentration of the alcohol solution, all of which fall within the scope of this application.

[0039] In some embodiments, in step S1, the collagen coating treatment involves incubating the filter membrane of the cell culture chamber with a coating solution containing collagen, so that collagen is adsorbed onto the surface of the filter membrane to form a collagen matrix layer. The coating solution contains collagen and an acidic solvent. The collagen remains soluble under acidic conditions. When the coating solution comes into contact with the filter membrane, collagen molecules gradually deposit on the filter membrane surface through hydrophobic interactions and electrostatic adsorption, forming a fibrous matrix layer. This incubation treatment provides a suitable extracellular matrix microenvironment for subsequent cell adhesion, promoting cell adhesion, spreading, and proliferation on the filter membrane. It is understood that those skilled in the art can appropriately adjust the concentration of collagen in the coating solution, the incubation temperature, and the time based on factors such as the filter membrane's material, pore size, and surface charge characteristics; all such adjustments fall within the scope of this application.

[0040] In some embodiments, the concentration of collagen in the coating solution is 2-5 mg / mL. In some embodiments, the collagen includes type I collagen, type III collagen, or a combination thereof. Preferably, the mass percentage of type I collagen is 90%-99% based on the total mass of collagen.

[0041] In some embodiments, the acidic solvent is a hydrochloric acid solution. In some embodiments, the incubation temperature is 4°C to 37°C, and the incubation time is 1 to 24 hours.

[0042] In some embodiments, the collagen coating treatment further includes rinsing the collagen-adsorbed filter membrane with a buffer solution after incubation to remove residual acidic solvent. Exemplarily, the buffer solution may be, but is not limited to, phosphate-buffered saline (PBS) or serum-free culture medium. The number of rinses may be, but is not limited to, 1 to 4 times.

[0043] In some embodiments, in step S2, the aforementioned primary epidermal keratinocytes are seeded in a cell suspension into the pretreated cell culture chamber. The concentration of the cell suspension is 1 × 10⁻⁶. 6 ~2×10 6A cell / mL concentration is beneficial for uniform distribution of cells on the filter membrane surface after seeding, promoting their proliferation and migration, while avoiding nutrient competition and excessive local accumulation due to excessive density. The seeding volume of the cell suspension can be determined by those skilled in the art based on the specifications of the selected cell culture chamber and the effective culture area. In some embodiments of this application, the seeding volume of the cell suspension can be 180~220 μL.

[0044] In some embodiments, the primary keratinocytes in step S2 are derived from human skin or mucous membrane tissue. These tissues can be obtained through tissue biopsy or purchased directly from commercial cell suppliers. The primary keratinocytes can be isolated using tissue block culture or enzymatic digestion methods (such as Dispase or trypsin digestion). In some specific embodiments of this application, the primary keratinocytes are derived from human epidermal tissue (such as trunk, limb skin, foreskin, etc.) or human lip tissue, as long as they can proliferate under in vitro culture conditions and form a non-keratinized stratified squamous epithelial structure; all such cell sources fall within the scope of cell sources applicable to this application.

[0045] In some embodiments, in step S2, the immersion culture medium used for the above-mentioned immersion culture includes 154 basal solution and also includes trisodium 2-phosphate-L-ascorbate. In some specific embodiments of this application, the above-mentioned immersion culture medium includes 154 basal solution and also includes 20~100 μg / mL of trisodium 2-phosphate-L-ascorbate.

[0046] In some embodiments, during step S2, the immersion culture medium is replaced with fresh medium every 2-3 days. Regular medium changes replenish the nutrients needed for cell growth and remove accumulated metabolic waste, thereby maintaining pH stability and nutrient supply in the culture system, which is beneficial for the continuous proliferation and healthy growth of keratinocytes. In some embodiments, the medium replacement sequence during the immersion culture process is as follows: first, aspirate the old medium from the lower chamber, then aspirate the old medium from the upper chamber; then, add fresh medium to the lower chamber first, and then add fresh medium to the upper chamber. This sequence prevents damage to the scaffold membrane or cell detachment due to excessive pressure difference between the inner and outer chambers.

[0047] In some embodiments, in step S2, the immersion culture time is 3 to 5 days. After this culture cycle, primary epidermal keratinocytes can form a confluent monolayer on the filter membrane surface and initially exhibit a layered structure, providing a suitable cell state for subsequent gas-liquid interface differentiation culture.

[0048] In some embodiments, in step S3, the conditions for the gas-liquid interface culture are as follows: epidermal differentiation culture medium is added to the lower chamber (i.e., outside the cell culture chamber) of the cell culture chamber, and no culture medium is added to the upper chamber (i.e., inside the cell culture chamber).

[0049] In some specific embodiments of this application, the above-mentioned gas-liquid interface culture steps are as follows: the cell culture chamber with preliminarily layered cells attached to the upper chamber is removed from the immersion culture system, the remaining culture medium in the cell culture chamber is aspirated, and then the cell culture chamber is transferred to a new culture plate (e.g., a 12-well plate). Epidermal differentiation culture medium is added to the lower chamber so that the liquid surface of the culture medium is rigidly contacted with the lower surface of the filter membrane. No culture medium is added to the upper chamber, so that the upper surface of the filter membrane remains exposed to the air, thereby effectively inducing the formation of non-keratinized stratified squamous epithelial structures. It is understood that the specific device for realizing gas-liquid interface culture is not limited to the above-mentioned 12-well plate. Other devices that can expose the upper surface of the filter membrane to air and contact the lower surface with liquid culture medium (e.g., culture plates of different specifications with corresponding lifting frames) can be used in this application and are all within the protection scope of this application.

[0050] In some embodiments, the epidermal differentiation culture medium includes a basal culture medium and further comprises one or more of the following: growth factor supplements, antioxidants, lipid supplements, β-adrenergic receptor agonists, amino acid supplements, and carrier proteins. The basal culture medium comprises a mixture of high-glucose DMEM medium and 154 basal culture medium; the growth factor supplement comprises HKGS (human keratinocyte growth supplement); the antioxidant comprises trisodium 2-phosphate-L-ascorbate; the lipid supplement comprises one or more of palmitic acid, oleic acid, linoleic acid, arachidonic acid, and dl-α-tocopheryl acetate; the β-adrenergic receptor agonist comprises isoproterenol hydrochloride; the amino acid supplement comprises L-serine and / or L-carnitine; and the carrier protein comprises fatty acid-free bovine serum albumin. The inventors of this application discovered during experiments that when the composition of the epidermal differentiation culture medium is unsuitable, the constructed model cannot form a non-keratinized squamous epithelial structure, resulting in numerous vacuoles or causing monolayer cell aging and thickening of the stratum corneum, thus failing to construct a reconstructed epidermal model.

[0051] In some specific embodiments of this application, the above-mentioned epidermal differentiation culture medium includes a basal culture medium in which the volume ratio of high glucose DMEM to 154 basal culture medium is 1:1, and also includes HKGS at a volume percentage of 0.5%~2%, 45~60 μg / mL of trisodium 2-phosphate-L-ascorbate, 5~15 μM isoproterenol hydrochloride, 8~12 mM L-serine, 8~12 μM L-carnitine, fatty acid-free bovine serum albumin (FAF-BSA) at a mass-volume percentage of 0.05%~0.2%, 20~30 μM palmitic acid, 20~30 μM oleic acid, 10~20 μM linoleic acid, 5~10 μM arachidonic acid, and 20~25 μM dl-α-tocopherol acetate.

[0052] In some embodiments, during the above-mentioned gas-liquid interface culture process, the above-mentioned epidermal differentiation culture medium is replaced with fresh medium every 1 to 2 days.

[0053] In some embodiments, the gas-liquid interface culture time is 3-7 days. The inventors of this application discovered in experiments that when using the aforementioned epidermal differentiation medium for gas-liquid interface culture, if the culture time is too long, it leads to aging of the monolayer cells and abnormal thickening of the stratum corneum. Ultimately, only a reconstructed epidermal model can be constructed, but a 3D lip-like epithelial model highly consistent with the structure of human lip tissue cannot be obtained. By strictly controlling the gas-liquid interface culture time, it is possible to ensure sufficient differentiation of the monolayer cells while effectively avoiding cell aging and excessive stratum corneum proliferation, thereby successfully constructing a three-dimensional epithelial model with realistic lip histological characteristics.

[0054] On the other hand, this application provides a 3D lip-like epithelial model, which is obtained by the above-described construction method.

[0055] The 3D lip-like epithelial model provided in this application consists of a basal layer, a spinous layer, and a non-keratinized squamous epithelial layer, without a typical granular layer. It is highly consistent with the structural characteristics of human lip tissue and can realistically simulate the physiological environment of lip skin.

[0056] Based on this, this application also provides a lip product irritation test kit, which includes the above-mentioned 3D lip-like epithelial model.

[0057] In some embodiments, the kit further includes a positive control, a negative control, a cell viability assay reagent, and an instruction manual. The positive control is selected from sodium dodecyl sulfate (SDS) or sodium dodecyl polyoxyethylene sulfate (SLS) at a concentration of 0.1%–0.2% (w / v). The negative control is sterile water or phosphate-buffered saline (PBS). By integrating a 3D lip-like epithelial model, controls, and assay reagents, this kit can be directly used for in vitro irritation testing of lip products and related compounds, offering convenient operation and stable results.

[0058] On the other hand, the histological features of the 3D lip-like epithelial model constructed in this application are highly consistent with the natural tissue of the human lip, and can more realistically reflect the effect of the analyte on the lip tissue. The advantages of this 3D lip-like epithelial model in terms of structural biomimicry and functional stability make it a potential application element in biosensors. Based on this, this application provides a reliable cell model foundation for the subsequent development of biosensors for rapid detection and real-time monitoring of lip skin irritation, and also opens up new technical paths for the further integration and automation of in vitro detection technologies.

[0059] Existing irritation testing methods are all based on conventional reconstructed epidermal models. Their testing logic and evaluation systems are designed for ordinary skin models, neglecting the unique structural characteristics of lip models. Direct application of these methods can easily lead to judgment bias. To address these shortcomings, this application also provides a lip product irritation testing method based on a 3D lip-like epithelial model, comprising the following steps: (1) The test sample was applied to the surface of the above-mentioned 3D lip-like epithelial model and cultured, and a negative control group and a positive control group were set up; (2) After incubation, the above 3D lip-like epithelial model was cleaned, cell viability was detected, and the relative cell viability of the test group relative to the negative control group was calculated; (3) Verify the validity of the data of the negative control group and the positive control group. If the verification is successful, determine the irritation of the lip product based on the relative cell viability of the test group to obtain the irritation type.

[0060] In some implementations, the incubation time in step (1) is 18-24 hours, and the incubation conditions are 37°C, 5% CO2, and 95% relative humidity, so that the irritant components in the test sample can fully penetrate and act on the 3D lip-like epithelial model. By setting up a negative control group (given a negative control) and a positive control group (given a positive control), the lower and upper limits of the irritation determination are respectively used to ensure the validity and comparability of the test.

[0061] In some embodiments, the cell viability detection method in step (2) includes the MTT assay, XTT assay, or ATP assay. The MTT assay reflects the number of viable cells by detecting the ability of mitochondrial dehydrogenases to reduce MTT to insoluble formazan crystals; the XTT assay is a modified version of the MTT assay, eliminating the need for a dissolution step for the soluble formazan product, making it simpler to operate; the ATP assay reflects cellular metabolic activity by detecting intracellular ATP content. In some preferred embodiments of this application, the MTT assay is used for cell viability detection. The formula for calculating relative cell viability is: Relative cell viability (%) = (Average OD value of the test group / Average OD value of the negative control group) × 100%.

[0062] In some implementations, to ensure the reliability and consistency of the test results in step (3), before determining the irritation level, it is necessary to confirm that the test data of the negative control group and the positive control group are within the valid range. Specifically, the tissue activity of the negative control group should not be lower than a preset threshold (e.g., the OD value should not be lower than 80% of the historical average of negative controls), and the relative cell viability of the positive control group should not be higher than 50%. Both conditions must be met for the test data to be considered valid; otherwise, the test must be repeated.

[0063] In some embodiments, the method for determining the irritation of the lip product in step (3) is as follows: if the relative cell viability of the test product is ≤50%, it is determined to be lip irritating; if the relative cell viability of the test product is >50%, it is determined to be non-irritating.

[0064] In some implementations, the above method further includes an inflammation risk assessment step: After the incubation culture was completed, the expression level of IL-1α in the culture medium used for incubation culture was detected; When the relative cell viability of the above test product is >50% and the IL-1α expression level is significantly higher than that of the negative control group ( p When the concentration is <0.05%, it indicates that the test sample has a potential risk of inflammatory reaction.

[0065] IL-1α is a pro-inflammatory cytokine released early by keratinocytes in response to exogenous stimulation. Upregulation of its expression level can reflect the degree of inflammatory response induced by the test substance. Even without a significant decrease in cell viability, elevated IL-1α levels can still serve as a sensitive indicator of potential inflammatory risk. When a test substance is determined to be non-irritating in the MTT assay but IL-1α expression is significantly elevated, it is recommended to conduct a comprehensive assessment of the lip safety of the test substance in conjunction with IL-1α test results, or to further conduct other supplementary tests (such as histological observation, detection of other inflammatory factors, etc.) to comprehensively determine the safety impact of the test substance on lip tissue.

[0066] It is understood that the expression level of IL-1α can be detected using conventional detection methods in the field, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, or real-time quantitative PCR. The detection materials can be culture supernatant (reflecting the level of IL-1α secreted extracellularly) or cell lysate (reflecting the total intracellular IL-1α level). Those skilled in the art can select appropriate detection methods and materials based on experimental conditions and detection sensitivity requirements, all of which fall within the scope of protection of this application.

[0067] This application also provides an application of the above-mentioned 3D lip-like epithelial model in the lip safety testing or efficacy testing of pharmaceuticals or cosmetics.

[0068] Specifically, the aforementioned safety testing includes risk assessments of irritation or inflammatory reactions for lip products (such as lipsticks, lip glosses, lip balms, lip care liquids, lip makeup removers, etc.) and compounds used on the lips (such as surfactants, preservatives, sunscreens, fragrances, and functional active ingredients, etc.). In safety testing applications, the test product can be applied to the surface of the aforementioned 3D lip-like epithelial model for cultivation. The safety impact of the test product on lip tissue can be assessed by detecting indicators such as model tissue viability (e.g., MTT assay) and inflammatory factor release (e.g., IL-1α). The 3D lip-like epithelial model provided in this application has tissue structure characteristics highly consistent with human lips, and its test results can more accurately reflect the safety risks of the test product in actual lip use scenarios, effectively avoiding false negatives or false positives caused by using conventional epithelial models.

[0069] The aforementioned efficacy testing includes efficacy verification and quantitative evaluation of lip products and their active ingredients that claim effects such as moisturizing, repairing, soothing, anti-inflammatory, or anti-aging. In efficacy testing applications, candidate active ingredients can be applied to the surface of the aforementioned 3D lip-like epithelial model, and changes in biomarkers related to enhanced lip barrier function, improved moisturizing ability, or inflammation relief can be detected, thereby providing scientific evidence for the product's efficacy claims.

[0070] It is understood that the above applications are not limited to the product types and testing indicators listed above. Any in vitro tests related to the lips that use the 3D lip-like epithelial model provided in this application to simulate the physiological characteristics of the human lip are within the scope of protection of this application.

[0071] It should be understood that materials of the same or similar type, model, quality, properties, or function as the reagents and instruments used in the following embodiments can be used to implement this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0072] The following are examples and comparative examples: Example 1 This embodiment provides a method for constructing a 3D lip-like epithelial model, including the following steps: (1) Tissue sampling and cell separation Primary epidermal keratinocytes were isolated from isolated skin tissue to construct a primary cell bank.

[0073] (2) In vitro expansion culture The isolated primary epidermal keratinocytes were expanded in vitro to form a primary keratinocyte bank.

[0074] (3) Pretreatment of cell culture chambers Cell culture chambers (i.e., Transwell chambers, with PET filter membranes purchased from Biosurfaces) were selected as cell support carriers. Before use, the cell culture chambers were pretreated: first, the chambers were pre-humidified with 50% USP-grade ethanol and cell culture-grade water (1.5 mL added to the lower chamber), and incubated at 37°C for 1 hour to remove air from the membrane pores. Then, the ethanol was discarded, and the lower and upper chambers were replaced with the same volume of sterile water, incubated at 37°C for 12 hours to prevent the filter membrane from drying out and allowing air ingress. Finally, the sterile water was removed, and then collagen coating solution (prepared by dissolving 90 wt% type I bovine collagen and 10 wt% type III bovine collagen in 0.012 N HCl to a total collagen concentration of 2 mg / mL) was added to the cell culture chambers, ensuring the collagen coating solution completely covered the filter membrane surface. The chambers were incubated at 37°C for 12 hours to allow collagen adsorption onto the filter membrane surface. After incubation, the remaining collagen working solution in the cell culture chamber was removed, and the chamber was rinsed with phosphate buffer. After rinsing three times, a cell culture chamber with a collagen matrix layer on the filter membrane surface was obtained, which is the pretreated cell culture chamber.

[0075] (4) Cell seeding and immersion culture The preparation concentration is 1×10 6 Primary keratinocyte suspension was prepared by adding 1 mL of epidermal immersion medium to the lower chamber of a pretreated cell culture chamber and 200 μL of the cell suspension to the upper chamber, ensuring the cell suspension evenly covered the filter membrane surface. After equilibration, 0.5 mL of epidermal immersion medium was added to the lower chamber. Immersion culture was carried out at 37°C and 5% CO2. Medium changes were started the day after inoculation, and then every 2 days thereafter. The medium change sequence was as follows: first, the old medium in the lower chamber was aspirated, then the old medium in the upper chamber was aspirated, then fresh epidermal immersion medium was added to the lower chamber, and then fresh epidermal immersion medium was added to the upper chamber. Immersion culture was carried out for 5 days until epidermal cells proliferated and formed a confluent monolayer. The formulation of the epidermal immersion medium was: 154 basal medium + 50 μg / mL trisodium 2-phosphate-L-ascorbate.

[0076] (5) Gas-liquid interface culture After immersion culture, the epidermal immersion medium was aspirated, and the cell culture chamber loaded with monolayer cells was transferred to a 12-well plate using an ALI-LC24 lifting rack. 0.6 mL of epidermal differentiation medium was added to the lower chamber, ensuring the medium level just touched the lower surface of the filter membrane. No medium was added to the upper chamber, leaving the upper surface of the filter membrane exposed to air. Differentiation culture at the gas-liquid interface was performed at 37°C, 5% CO2, and 95% humidity, with daily medium changes. After 7 days of gas-liquid interface culture, a 3D model was formed. The epidermal differentiation medium was formulated as follows: a basal medium consisting of high-glucose DMEM and 154 basal medium in a 1:1 volume ratio, and 1% (v / v) HKGS, 50 μg / mL trisodium 2-phosphate-L-ascorbate, 10 μM isoproterenol hydrochloride, 10 mM L-serine, 10 mM L-carnitine, 0.1% (w / v) fatty acid-free bovine serum albumin (FAF-BSA), 25 μM palmitic acid, 25 μM oleic acid, 15 μM linoleic acid, 7 μM arachidonic acid, and 22 μM dl-α-tocopherol acetate.

[0077] Histological section analysis of the 3D model constructed from the aforementioned primary keratinocyte bank was performed using H&E staining. The results showed that the model constructed in this embodiment consisted of the basal layer, spinous layer, and non-keratinized squamous epithelium, and lacked a typical granular layer, exhibiting a high degree of consistency with the tissue structure of the human lip. Figure 2 ).

[0078] Example 2 The method for constructing the 3D lip-like epithelial model provided in this embodiment refers to the method provided in Embodiment 1, including tissue sampling and cell separation, in vitro expansion culture, cell culture chamber pretreatment, cell seeding and immersion culture, and gas-liquid interface differentiation culture. The gas-liquid interface culture time is 3 days and 5 days, respectively, and the groups with different culture times are treated with epidermal differentiation medium 1 and epidermal differentiation medium 2, respectively.

[0079] The formulation of epidermal differentiation medium 1 is as follows: a basal medium composed of high glucose DMEM and 154 basal medium in a volume ratio of 1:1, and 1% (v / v) HKGS, 45 μg / mL trisodium 2-phosphate-L-ascorbate, 15 μM isoproterenol hydrochloride, 8 mM L-serine, 8 mM L-carnitine, 0.2% (w / v) fatty acid-free bovine serum albumin (FAF-BSA), 30 μM palmitic acid, 20 μM oleic acid, 20 μM linoleic acid, 10 μM arachidonic acid, and 20 μM dl-α-tocopherol acetate.

[0080] The formulation of epidermal differentiation medium 2 is as follows: a basal medium composed of high glucose DMEM and 154 basal medium in a volume ratio of 1:1, and 1% (v / v) HKGS, 60 μg / mL trisodium 2-phosphate-L-ascorbate, 5 μM isoproterenol hydrochloride, 12 mM L-serine, 12 mM L-carnitine, 0.1% (w / v) fatty acid-free bovine serum albumin (FAF-BSA), 20 μM palmitic acid, 30 μM oleic acid, 10 μM linoleic acid, 5 μM arachidonic acid, and 25 μM dl-α-tocopherol acetate.

[0081] Comparative Example 1 The method for constructing the 3D model provided in this comparative example is the same as that in Example 1, except that the gas-liquid interface culture time is 9 days.

[0082] Histological section analysis of the 3D model constructed in this comparative example was performed using H&E staining. The results showed that the stratum corneum of the model constructed in this comparative example was abnormally thickened, and ultimately only a reconstructed epidermal model could be constructed. Figure 3 ).

[0083] Comparative Example 2 The 3D model provided in this comparative example is constructed using the same method as in Example 1, except that the epidermal differentiation medium formulation is: a basal medium composed of high-glucose DMEM and 154 basal medium in a 1:1 volume ratio, and 1% (v / v) HKGS, 100 μg / mL trisodium 2-phosphate-L-ascorbate, 10 μM isoproterenol hydrochloride, 15 mM L-serine, 15 mM L-carnitine, 0.1% (w / v) fatty acid-free bovine serum albumin (FAF-BSA), 15 μM palmitic acid, 15 μM oleic acid, 15 μM linoleic acid, 10 μM arachidonic acid, and 25 μM dl-α-tocopherol acetate.

[0084] Histological section analysis of the 3D model constructed in this comparative example was performed using H&E staining. The results showed that the model constructed in this comparative example contained numerous vacuoles. Figure 4 It is impossible to construct a 3D lip-like epithelial model.

[0085] Example 3 This embodiment provides a method for testing the irritation of lip products based on a 3D lip-like epithelial model, including the following steps: (1) The above-mentioned 3D lip epithelial model was transferred from solid culture medium to a 6-well plate containing model culture medium (DMEM) and incubated at 37°C and 5% CO2 for 18 hours for recovery. Then, different types of lip test samples (including positive control, negative control, solvent control, common surfactants, sensory irritants, acidic irritants, chemical sunscreens, lip gloss products, and makeup removers) were applied to the surface of the 3D lip epithelial model with 30µL of sample (liquid) or 30mg of sample (solid). Fresh model culture medium was then replaced, and the samples were incubated at 37°C and 5% CO2 for another 18 hours to simulate the actual use scenario of lip products. The specific information of the test samples is shown in Table 1. Among them, the simulated suspected positive lip gloss product group: lip gloss + X% menthol, that is, X% (w / w) menthol was added to the lip gloss stock solution, mixed well, and used directly. Simulated suspected positive makeup remover product group: makeup remover gel + 1% salicylic acid, that is, add 1% (w / w) salicylic acid to the makeup remover gel concentrate, mix well, and then dilute with solvent until the mass fraction of the makeup remover gel concentrate in the final solution is 5%.

[0086] (2) After incubation, the residual test samples on the model surface were cleaned, and cell viability was detected by the MTT assay: the model was co-incubated with MTT reagent for 4 h, and then 1,3-butanediol was added to dissolve the blue-purple formazan crystals generated in the cells. The absorbance value was measured at a wavelength of 490 nm. Then, the cell viability of each test group relative to the negative control group was calculated according to the following formula: relative cell viability (%) = (average OD value of test group / average OD value of negative control group) × 100%.

[0087] (3) Lip irritation was determined based on the relative cell viability of the test groups: if the relative cell viability was ≤50%, it was determined to be lip irritating; if the relative cell viability was >50%, it was determined to be non-irritating. The determination results are shown in Table 1. Figure 5 As shown.

[0088] Table 1. Lip test samples

[0089] The results showed that the negative control (ddH2O) and solvent control (1,3-butanediol) were both non-irritating, as expected. 0.2% SDS, sodium lauryl ether sulfate, ammonium lauryl ether sulfate, and lactic acid complexes with concentrations above 5% all exhibited significant irritation in the lip model (relative cell viability <10%), while capsaicin, capsaicin, menthol, ethylhexyl methoxycinnamate, lip gloss products (with or without menthol), and makeup remover gel products showed no irritation at the 5% test concentration. These results fully demonstrate that the lip product irritation testing method based on a 3D lip-like epithelial model has excellent discriminative ability and applicability for different types of lip-related samples.

[0090] The test results of 0.2% SDS on a 3D lip-like epithelial model in this embodiment are compared with those of existing technologies. References ( Journal of Cosmetic Dermatology The study published in 2021, 20(6), 1933-1941, on the application of in vitro recombinant human skin models in cosmetic irritation testing showed that 0.2% SLS (i.e., 0.2% SDS) showed no irritation (relative cell viability > 50%) after being applied to a conventional recombinant epidermal model. However, in this embodiment, 0.2% SDS showed significant irritation (relative cell viability < 5%) on the 3D lip-like epithelial model constructed in this application. The above comparative results indicate that the detection sensitivity of the 3D lip-like epithelial model used in this application is significantly higher than that of the conventional recombinant epidermal model, and it can effectively identify low-concentration irritating components that cannot be detected by the conventional model, thereby avoiding false negatives that may occur in the irritation assessment of lip products and effectively making up for the lack of adaptability of the conventional recombinant epidermal model to the specific lip scenario.

[0091] (4) After incubation, the model culture medium of the above sensory stimulant test groups (capsaicin, menthol) was collected, and the expression level of IL-1α was detected by ELISA. When the relative cell viability of the test sample was >50% (i.e., it was judged to be non-irritating) but the IL-1α expression level was significantly higher than that of the negative control group ( p A value <0.05 indicates that although the test sample did not cause significant cytotoxic damage, it still carries a potential risk of inflammatory response. Test results are as follows... Figure 6 As shown.

[0092] It can be seen that the capsaicin-treated group (test concentration of 0.05% and above) was determined to be non-irritating in the MTT assay, but the IL-1α expression level in its 3D lip-like epithelial model was significantly higher than that in the negative control group. p The result (<0.05) suggests that the lip model has a potential risk of inflammatory response at this capsaicin exposure concentration.

[0093] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for constructing a 3D lip-like epithelial model, characterized in that, Includes the following steps: S1. The cell culture chambers are sequentially subjected to hydrophilic treatment, sterile water incubation, and collagen coating treatment to obtain pretreated cell culture chambers; S2. Primary epidermal keratinocytes are seeded into the pretreated cell culture chamber for immersion culture, allowing the cells to proliferate on the filter membrane of the cell culture chamber to form a confluent monolayer of cells. S3. The cell culture chamber loaded with the monolayer cells is subjected to gas-liquid interface culture to allow the monolayer cells to differentiate into a 3D model with a non-keratinized stratified squamous epithelial structure, thus obtaining the 3D lip-like epithelial model.

2. The construction method according to claim 1, characterized in that, In step S2, the primary epidermal keratinocytes are seeded in the pretreated cell culture chamber in the form of a cell suspension, and the concentration of the cell suspension is 1×10⁻⁶. 6 ~2×10 6 pcs / mL; and / or, The immersion culture uses a 154 basal medium, which also includes trisodium 2-phosphate-L-ascorbic acid; and / or, During the immersion culture process, the immersion medium should be replaced with fresh medium every 2-3 days; and / or, The immersion culture time is 3 to 5 days.

3. The construction method according to claim 1, characterized in that, In step S3, the conditions for gas-liquid interface culture are as follows: epidermal differentiation culture medium is added to the lower chamber of the cell culture chamber, and no culture medium is added to the upper chamber of the cell culture chamber. During the gas-liquid interface culture process, the epidermal differentiation culture medium is replaced with fresh medium every 1 to 2 days. The gas-liquid interface culture time is 3-7 days; Preferably, the epidermal differentiation culture medium includes a basal culture medium and further comprises one or more of the following: growth factor supplements, antioxidants, lipid supplements, β-adrenergic receptor agonists, amino acid supplements, and carrier proteins. The basal culture medium comprises a mixture of high-glucose DMEM medium and 154 basal culture medium; The growth factor supplement contains HKGS; The antioxidant comprises trisodium 2-phosphate-L-ascorbic acid; The lipid supplement contains one or more of palmitic acid, oleic acid, linoleic acid, arachidonic acid, and dl-α-tocopherol acetate; The β-adrenergic receptor agonist includes isoproterenol hydrochloride; The amino acid supplement contains L-serine and / or L-carnitine; The carrier protein contains bovine serum albumin.

4. The construction method according to claim 3, characterized in that, The epidermal differentiation culture medium contains the following components: A basal medium with a volume ratio of 1:1 between high-glucose DMEM medium and 154 basal medium; HKGS with a volume percentage of 0.5% to 2%; Trisodium 2-phosphate-L-ascorbic acid at a concentration of 45~60 μg / mL; Isoproterenol hydrochloride at a concentration of 5-15 μM; L-serine at a concentration of 8-12 mM; L-carnitine at a concentration of 8-12 μM; Fatty acid-free bovine serum albumin with a mass-volume percentage of 0.05%~0.2%; Palmitic acid at a concentration of 20-30 μM; Oleic acid at a concentration of 20~30μM; Linoleic acid at a concentration of 10~20μM; Arachidonic acid at a concentration of 5-10 μM; and dl-α-tocopherol acetate at a concentration of 20-25 μM.

5. A 3D lip-like epithelial model, characterized in that, It is obtained by the construction method described in any one of claims 1 to 4.

6. A lip product irritation test kit, characterized in that, Includes the 3D lip-like epithelial model as described in claim 5.

7. A method for testing the irritation of lip products based on a 3D lip-like epithelial model, characterized in that, Includes the following steps: (1) The test sample was applied to the surface of the 3D lip-like epithelial model and incubated, and a negative control group and a positive control group were set up; (2) After incubation, the 3D lip-like epithelial model was cleaned, cell viability was detected, and the relative cell viability of the test group relative to the negative control group was calculated; (3) Verify the validity of the data of the negative control group and the positive control group. If the verification is successful, determine the irritation of the lip product based on the relative cell viability of the test group to obtain the irritation type.

8. The method for testing the irritation of lip products according to claim 7, characterized in that, The incubation period in step (1) is 18-24 hours; The cell viability detection methods described in step (2) include the MTT assay, XTT assay, or ATP assay; The method for determining lip irritation in step (3) is as follows: if the relative cell viability of the test product is ≤50%, it is determined to be lip irritating; if the relative cell viability of the test product is >50%, it is determined to be non-irritating.

9. The method for testing the irritation of lip products according to claim 7, characterized in that, It also includes an inflammation risk assessment step: After the incubation culture was completed, the expression level of IL-1α in the culture medium used for incubation culture was detected; When the relative cell viability of the test product is >50% and the IL-1α expression level is significantly higher than that of the negative control group, it suggests that the test product has a potential risk of inflammatory response.

10. The application of a 3D lip-like epithelial model as described in claim 5 in the lip safety or efficacy testing of pharmaceuticals or cosmetics.

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