CN113293126B

CN113293126B - In-vitro construction method of human vaginal mucosa model

Info

Publication number: CN113293126B
Application number: CN202110356820.4A
Authority: CN
Inventors: 李润芝; 卢永波; 张勇杰; 李潇; 杨文娟
Original assignee: Guangdong Biocell Biotechnology Co ltd
Current assignee: Guangdong Biocell Biotechnology Co ltd
Priority date: 2021-04-01
Filing date: 2021-04-01
Publication date: 2023-11-07
Anticipated expiration: 2041-04-01
Also published as: CN113862214A; CN113293126A

Abstract

The application provides an in vitro construction method of a human vaginal mucosa model, which adopts vaginal fibroblasts and collagen to construct a matrix layer, and inoculates vaginal epithelial cells on the basis to form an in vitro vaginal mucosa model with a double-layer structure, which is similar to natural tissues in structure, and the vaginal irritation experiment of the product on the model is closer to the in vivo real condition, and the experimental result is more accurate and reliable. The sectional culture method can ensure the nutrition requirements of cells in different development stages, is beneficial to the formation of a multi-layered vaginal mucosa epithelial structure, and can shorten the construction time and reduce the production cost.

Description

In-vitro construction method of human vaginal mucosa model

Technical Field

The application belongs to the field of biomedicine, and particularly relates to an in-vitro construction method of a human vaginal mucosa model.

Background

The human vaginal mucosa consists of thick, non-keratinized, stratified squamous epithelium, rich in glycogen, and small numbers of other types of cells, such as macrophages and langerhans cells. Below the epithelial cells is the lamina propria, comprising a number of elastic fibers and a dense network of blood vessels, so the vaginal mucosa is an excellent route to deliver drugs for local and systemic treatment. However, due to prolonged exposure to various pathogens within the lumen of the genital tract, vaginal epithelium is also a potential site for many pathogens to enter the body, for example, following use of feminine care and cosmetic products, contraceptive or microbiocides, may cause mild injury, which may cause tissue irritation and render the vaginal epithelium particularly susceptible to various types of infections. Formulations specifically formulated for the vaginal mucosa of humans, such as pharmaceuticals, cosmetics and personal care products, can sometimes cause adverse local or systemic side effects. Therefore, assessing the compatibility of newly developed cosmetics, personal care products or topically applied drugs with human vaginal mucosal surfaces prior to product release is a critical issue that manufacturers need to address.

The stratified differentiated human vaginal epithelial model has the potential to overcome certain drawbacks of the cell monolayer membrane, as the former comprises a barrier layer and allows topical application of active ingredients and final formulations, including those that are not water soluble. In addition to avoiding animal welfare risk and species variability problems, in vitro tissue models can often distinguish very mild products to which animal models are insensitive.

The RHVE model of SkinEthic company in France adopts a vulva epidermoid carcinoma cell line A431 as seed cells, the cell line is stable and reliable, the problem of difficult source of seed cells can be solved, but the RHVE model only comprises an epithelial layer consisting of A431 cells, lacks stroma layer cells and stroma, and therefore, the tissue structure is greatly different from that of natural vaginal tissue. The tissue structure of the EpiVaginal vaginal model of MatTek company in the United states and the human vaginal epithelium 3D model disclosed in Chinese patent CN201710048227.7 are highly similar to that of natural vaginal mucosa, however, due to the limited sources of available vaginal tissue and the fact that the vaginal mucosa epithelium is a multi-layer squamous epithelium, most cells belong to differentiated mature cells, the proliferation capacity is low, and only a small part of basal layer cells have strong proliferation capacity, so that the requirement of in vitro mass expansion culture conditions is high, the in vitro mass production cannot be realized, and the market demand is met. The human vaginal epithelium 3D model disclosed in Chinese patent CN201710048227.7 has some defects, such as poor model layering and weak barrier function caused by a single culture system which cannot adapt to the nutritional requirements of different stages of cells, and long in-vitro model construction time, a batch of models need to be constructed for one month, and the overlong production period can increase uncontrollable factors in the process to influence the stability of the model, so that the model is only limited to laboratory culture and is not industrially produced at present, and no commercial human vaginal mucosa model exists at home.

Disclosure of Invention

Aiming at the problems existing in the prior art, the application provides an in-vitro construction method of a human vaginal mucosa model.

In order to achieve the aim of the application, the application is realized by adopting the following technical scheme:

an in vitro construction method of a human vaginal mucosa model comprises the following steps:

A. construction of a matrix layer containing fibroblasts:

(1) Taking vaginal fibroblasts of 5 th to 10 th generation, and preparing a cell suspension by using DMEM (DMEM) containing 20% fetal bovine serum;

(2) Uniformly mixing the cell suspension and the neutral collagen scaffold according to the volume ratio of 1:9, inoculating the mixture into a cell culture chamber for first culture, then respectively adding HVE-1 culture solution into the interior and the exterior of the chamber, and forming a matrix layer in a vaginal mucosa model after secondary culture;

the HVE-1 culture solution is prepared according to the following method: taking a culture medium mixed by DMEM and F12 according to a volume ratio of 1-3:1 as a base solution, adding 10% fetal bovine serum into the base solution, adding 2.0-6.0 mu mol/mL glutamine, 15-30 mu g/mL adenine, 5-15 mu g/mL insulin, 0.5-2.0 mu g/mL hydrocortisone, 25-100 mu g/mL vitamin C, 1-10 ng/mL fibroblast growth factor and 5-20 ng/mL transferrin;

B. inoculation of cells:

sucking the HVE-1 culture solution on the surface of the matrix layer, preparing the vaginal epithelial cells or vulva epidermoid carcinoma cell line A431 cells into a cell suspension by using the HVE-2 culture solution, inoculating the cell suspension onto the matrix layer in a cell culture chamber, and replacing the culture medium with the HVE-2 culture solution for submerged culture;

the HVE-2 culture solution is prepared according to the following method: adding 5% fetal bovine serum, 2.0-6.0 mu mol/mL glutamine, 15-30 mu g/mL adenine, 5-15 mu g/mL insulin, 0.5-2.0 mu g/mL hydrocortisone, 1-10 ng/mL fibroblast growth factor, 5-20 ng/mL transferrin, 0.5-2.0 ng/mL epidermal growth factor, 0.1-0.4 nmol/mL triiodothyronine and 5-10 nmol/mL isoprenaline into a culture medium mixed by DMEM and F12 according to a volume ratio of 1-3:1 serving as a base solution;

C. culture of vaginal mucosa model:

sucking the HVE-2 culture solution on the surface of the culture, and lifting the cell culture chamber to an air-liquid interface for gas-liquid surface culture; and replacing the culture medium with HVE-3 culture solution, and culturing to obtain the double-layer vaginal mucosa model. The double-layer vaginal mucosa model is a double-layer vaginal mucosa model with a matrix layer containing fibroblasts and an epithelial layer containing vaginal epithelial cells.

The HVE-3 culture solution is prepared according to the following method: the culture medium mixed by DMEM and F12 according to the volume ratio of 1-3:1 is taken as a basic solution, 5% fetal bovine serum, 2.0-6.0 mu mol/mL glutamine, 15-30 mu g/mL adenine, 5-15 mu g/mL insulin, 0.5-2.0 mu g/mL hydrocortisone, 1-10 ng/mL fibroblast growth factor, 5-20 ng/mL transferrin, 2.0-5.0 ng/mL epidermal growth factor, 0.1-0.4 nmol/mL triiodothyronine, 5-10 nmol/mL isoprenaline, 25-100 mu g/mL vitamin C and 1.0-1.5 mu mol/mL CaCl are added into the basic solution ₂ 。

Preferably, the steps of isolation and culture of vaginal fibroblasts in step a are as follows:

(1) Placing the separated vaginal tissue lamina propria into collagenase digestive juice for digestion, stopping digestion by using DMEM culture solution containing 10% fetal calf serum, filtering, centrifuging to remove supernatant, and collecting vaginal fibroblasts;

(2) The vaginal fibroblasts were washed with PBS, resuspended cells were pelleted in DMEM medium containing 10% new born calf serum, and subcultured until the fifth generation for the construction of the matrix layer in the vaginal mucosa model.

Preferably, the steps of isolation and culture of vaginal epithelial cells in step B are as follows:

(1) Placing the vaginal tissue of the human body in a culture dish, washing the vaginal tissue with PBS solution for 6 times, removing submucosal tissue, cutting the tissue block into pieces, and adding into dispersive enzyme for digestion;

(2) Separating out the vaginal mucosa epithelial layer and the lamina propria by using ophthalmic forceps, and putting the vaginal mucosa epithelial layer into pancreatin-EDTA for digestion;

(3) Stopping digestion by using DMEM culture solution containing 10% of fetal calf serum, centrifuging to remove supernatant after filtering, and collecting vaginal mucosal epithelial cells;

(4) And (3) washing vaginal mucosa epithelial cells by PBS, and precipitating the resuspended cells in serum-free epithelial cell culture solution to obtain vaginal epithelial cell suspension separated into single cells, and subculturing until the second generation to obtain the vaginal epithelial cells with the purity of more than 95%.

Preferably, the serum-free epithelial cell culture medium in the step (4) in the step A is a culture medium based on a mixture of DMEM and F12 in a volume ratio of 1-3:1, wherein 2.0-6.0 mu mol/mL of glutamine, 2.0-5.0 ng/mL of epidermal growth factor, 15-30 mu g/mL of bovine pituitary extract, 10-15 ng/mL of insulin, 0.5-2.0 mu g/mL of hydrocortisone and 0.1-0.4 mu mol/mL of CaCl are added ₂ 。

The serum-free epithelial cell culture fluid in the application takes DMEM/F12 as a base fluid, contains factors and proteins with proper concentrations such as glutamine, bovine Pituitary Extract (BPE), epidermal Growth Factor (EGF), insulin, hydrocortisone, calcium chloride and the like, and is suitable for culturing vaginal epithelial cells, including primary cells extracted from normal tissues and various immortalized and transformed vaginal epithelial cell lines. The serum-free epithelial cell culture solution of the application contains no serum component, so that the pollution of fibroblasts in the process of extracting vaginal epithelial cells can be effectively reduced, and the purity of the vaginal epithelial cells in the second generation can reach more than 95 percent after subculturing. In addition, the method is also suitable for culturing various immortalized and transformed vaginal epithelial cell lines, is favorable for large-scale production, and can well save labor and material cost.

Preferably, the culturing process of the exocrine epidermoid carcinoma cell line a431 cells in step B is: the re-suspended vulva epidermoid carcinoma cell line A431 cells are deposited in DMEM culture solution containing 10% fetal calf serum, 2.0-6.0 mu mol/mL glutamine, 0.1-1 mu mol/mL sodium pyruvate and 4.5g/L high sugar, and are used for constructing epithelial layer after subculture until the seventh generation.

The double-layer vaginal mucosa model constructed by the cell culture of the vulva epidermoid carcinoma cell line A431 is a double-layer vaginal mucosa model with a matrix layer containing fibroblasts and an epithelial layer containing the vulva epidermoid carcinoma cell line A431.

Preferably, the preparation method of the neutral collagen scaffold in the step A comprises the following steps: weighing collagen, placing the collagen into 0.1% acetic acid solution to prepare 4-10 mg/mL collagen solution, placing the collagen solution on ice after complete dissolution, uniformly mixing the collagen solution and 10% fetal bovine serum DEME culture solution according to the volume ratio of 1:8, and adding 0.1M NaOH solution to adjust the pH to 7.2-7.4.

Preferably, the primary culture temperature in the step A is 37 ℃, the primary culture time is 2-3 hours, the secondary culture temperature is 37 ℃, and the secondary culture time is 2-4 days.

Preferably, the incubation temperature in step B is 37℃and the incubation time is 4 days.

Preferably, the culture temperature in step C is 37℃and the culture time is 8 to 12 days.

Preferably, the PBS solution contains 100U/mL penicillin and 100U/mL streptomycin.

Preferably, the EDTA concentration is 0.2mg/mL.

Preferably, in the step C, glacial acetic acid is diluted into 0.1% acetic acid solution by deionized water, filtered and sterilized for use.

Preferably, the seed cells used in the construction of the vaginal mucosal epithelium in the present application are one or more of the normal primary vaginal epithelial cells, the immortalized vaginal epithelial cell lines Ect1, E6E7, end1, E6E7, vk2, E6E7 or the vulvar epidermoid carcinoma cell line a 431. The seed cells are selected variously, the in vitro construction repeatability is good, the problems of difficult source of the seed cells, poor cell stability and the like are solved, and the industrialized preparation can be realized.

Compared with the prior art, the application has the beneficial effects that:

the application discloses an in vitro construction method of a human vaginal mucosa model, which adopts vaginal fibroblasts and collagen to construct a matrix layer, and inoculates vaginal epithelial cells on the basis to form an in vitro vaginal mucosa model with a double-layer structure, which is similar to natural tissues in structure, and the vaginal irritation experiment of the product on the model is closer to the in vivo real condition, and the experimental result is more accurate and reliable. The application adopts two stages of submerged culture and air-liquid surface culture when co-culturing vaginal epithelial cells and matrix layers, and the nutrient components of the culture solution are finely regulated according to different requirements of cell proliferation and differentiation in different culture stages.

Drawings

FIG. 1A is an apparent picture of a bilayer vaginal mucosal epithelium model constructed using normal human vaginal epithelium cell culture in accordance with the present application;

FIG. 1B is a histological H & E staining photograph of a model of double-layer vaginal mucosa epithelium constructed using normal human vaginal epithelium cell culture in accordance with the present application;

FIG. 2A is an apparent photograph of a bilayer vaginal mucosal epithelium model constructed using a vulvar epidermoid carcinoma cell line A431 cell culture in accordance with the present application;

FIG. 2B is a histological H & E staining photograph of a model of double-layer vaginal mucosa epithelium constructed using cell culture of vulvar epidermoid carcinoma cell line A431 in accordance with the present application.

Detailed Description

The technical scheme of the application is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the application and are not to be construed as a specific limitation thereof.

Example 1

A. isolation and culture of vaginal epithelial cells:

(1) Placing human vaginal tissue in a culture dish, cleaning with PBS solution precooled at 4deg.C for 6 times, removing submucosal tissue, cutting tissue block into 0.2cm×0.2cm size, adding into 1.5U/mL of dispersing enzyme, and digesting at 4deg.C overnight;

(2) Separating out the vaginal mucosa epithelial layer and the lamina propria by using ophthalmic forceps, putting the vaginal mucosa epithelial layer into 0.25% pancreatin-EDTA, and digesting for 30min at 37 ℃, wherein the concentration of EDTA is 0.2mg/mL;

(3) Stopping digestion with DMEM culture solution containing 10% fetal bovine serum, filtering with 200 mesh sieve, centrifuging to remove supernatant, and collecting vaginal mucosal epithelial cells;

(4) Washing vaginal mucosa epithelial cells with PBS, precipitating the resuspended cells in serum-free epithelial cell culture solution to obtain vaginal epithelial cell suspension separated into single cells, inoculating into T75 culture flask, culturing at 37deg.C, and 5% CO ₂ Culturing in an incubator, and subculturing until the second generation to obtain vaginal epithelial cells with purity of more than 95%;

the serum-free epithelial cell culture solution is prepared according to the following method: a culture medium in which DMEM and F12 were mixed at a volume ratio of 1:1 was used as a base solution, to which 2.0. Mu. Mol/mL of glutamine, 2.0ng/mL of EGF, 15. Mu. G/mL of BPE,10ng/mL of insulin, 0.5. Mu. G/mL of hydrocortisone, 0.1. Mu. Mol/mL of CaCl were added ₂ 。

B. Isolation and culture of vaginal fibroblasts:

(1) Placing the separated vaginal tissue lamina propria into 200U/mL collagenase digestion solution, digesting for 3 hours at 37 ℃, stopping digestion by using DMEM culture solution containing 10% fetal bovine serum, filtering by a 200-mesh screen, centrifuging to remove the supernatant, and collecting vaginal fibroblasts;

(2) Washing vaginal fibroblasts with PBS, precipitating the resuspended cells in DMEM culture solution containing 10% new born calf serum, inoculating into T75 culture flask, culturing at 37deg.C, 5% CO ₂ Subculturing under the condition until the fifth generation, and constructing a matrix layer in the vaginal mucosa model;

C. preparation of a neutral collagen scaffold:

weighing collagen, placing the collagen into 0.1% acetic acid solution to prepare 4mg/mL collagen solution, placing the collagen solution on ice after complete dissolution, uniformly mixing the collagen solution and 10% fetal bovine serum DEME culture solution according to the volume ratio of 1:8, and adding 0.1M NaOH solution to adjust the pH to 7.2;

D. construction of a matrix layer containing fibroblasts:

(1) Vaginal fibroblasts of the 5 th generation were prepared to a density of 2X 10 with DMEM containing 20% fetal bovine serum ⁶ Cell suspension per mL;

(2) Mixing cell suspension and neutral collagen scaffold at volume ratio of 1:9, inoculating into Transwell chamber, culturing at 37deg.C for 2 hr, andthen HVE-1 culture solution is added into the interior and exterior of Transwell chamber respectively, 37 deg.C and 5% CO ₂ Culturing in an incubator for 2 days to form a matrix layer in the vaginal mucosa model;

the HVE-1 culture solution is prepared according to the following method: a culture medium in which DMEM and F12 were mixed at a volume ratio of 1:1 was used as a base solution, to which 10% fetal bovine serum, 2.0. Mu. Mol/mL glutamine, 15. Mu.g/mL adenine, 5. Mu.g/mL insulin, 0.5. Mu.g/mL hydrocortisone, 25. Mu.g/mL vitamin C,1ng/mL fibroblast growth factor, and 5ng/mL transferrin were added.

E. Inoculation of vaginal epithelial cells:

(1) Sucking the culture medium on the surface of the matrix layer, and preparing the obtained vaginal epithelial cells or A431 cell line into 2.0X10 by using HVE-2 culture solution ⁶ Inoculating a cell suspension to a substrate layer in a Transwell chamber;

(2) The culture medium is replaced by HVE-2 culture solution for submerged culture at 37deg.C with 5% CO ₂ Culturing in an incubator for 4 days;

the HVE-2 culture solution is prepared according to the following method: a culture medium in which DMEM and F12 were mixed at a volume ratio of 1:1 was used as a base solution, to which 5% fetal bovine serum, 2.0. Mu. Mol/mL glutamine, 15. Mu.g/mL adenine, 5. Mu.g/mL insulin, 0.5. Mu.g/mL hydrocortisone, 1ng/mL fibroblast growth factor, 5ng/mL transferrin, 0.5ng/mL EGF,0.1nmol/mL triiodothyronine, 5nmol/mL isoprenaline were added.

F. Culture of vaginal mucosa model:

(1) Sucking the HVE-2 culture solution on the surface of the culture in the Transwell chamber, and lifting the Transwell chamber to an air-liquid interface for gas-liquid surface culture;

(2) The HVE-2 culture solution was replaced with HVE-3 culture solution at 37deg.C with 5% CO ₂ Culturing in an incubator for 8 days to obtain a double-layer vaginal mucosa model with a matrix layer containing fibroblasts and an epithelial layer containing vaginal epithelial cells.

The HVE-3 culture solution is prepared according to the following method: the culture medium mixed by DMEM and F12 according to the volume ratio of 1:1 is taken as a base solution, and 5% of fetus is added into the base solutionBovine serum, 2.0. Mu. Mol/mL glutamine, 15. Mu.g/mL adenine, 5. Mu.g/mL insulin, 0.5. Mu.g/mL hydrocortisone, 1ng/mL fibroblast growth factor, 5ng/mL transferrin, 2.0ng/mL EGF,0.1nmol/mL triiodothyronine, 5nmol/mL isoprenaline, 25. Mu.g/mL vitamin C, 1.0. Mu. Mol/mL CaCl ₂ 。

Example 2

A. isolation and culture of vaginal epithelial cells:

(2) Separating out the vaginal mucosa epithelial layer and the lamina propria by using ophthalmic forceps, putting the vaginal mucosa epithelial layer into 0.25% pancreatin-EDTA, and digesting for 45min at 37 ℃, wherein the concentration of the EDTA is 0.2mg/mL;

the serum-free epithelial cell culture solution is prepared according to the following method: DMEM and F12 in a volume ratio of 2:1, to which 4.0. Mu. Mol/mL glutamine, 3.0ng/mL EGF, 20. Mu.g/mL BPE,13ng/mL insulin, 1.5. Mu.g/mL hydrocortisone, 0.2. Mu. Mol/mL CaCl were added ₂ ；

B. Isolation and culture of vaginal fibroblasts:

C. preparation of a neutral collagen scaffold:

weighing collagen, placing the collagen into 0.1% acetic acid solution to prepare 6mg/mL collagen solution, placing the collagen solution on ice after complete dissolution, uniformly mixing the collagen solution and 10% fetal bovine serum DEME culture solution according to the volume ratio of 1:8, and adding 0.1M NaOH solution to adjust the pH to 7.3;

D. construction of a matrix layer containing fibroblasts:

(1) Vaginal fibroblasts of the 8 th generation were prepared to a density of 5X 10 with DMEM containing 20% fetal bovine serum ⁶ Cell suspension per mL;

(2) Uniformly mixing a cell suspension and a neutral collagen scaffold according to a volume ratio of 1:9, inoculating into a Transwell chamber, culturing for 2.5 hours at 37 ℃, and then respectively adding HVE-1 culture solution, 37 ℃ and 5% CO into the inside and the outside of the Transwell chamber ₂ Culturing in an incubator for 3 days to form a matrix layer in the vaginal mucosa model;

the HVE-1 culture solution is prepared according to the following method: adding 10% fetal bovine serum, 4.0 mu mol/mL glutamine, 20 mu g/mL adenine, 10 mu g/mL insulin, 1.2 mu g/mL hydrocortisone, 75 mu g/mL vitamin C,5ng/mL fibroblast growth factor, 15ng/mL transferrin to a base solution of a culture medium mixed by DMEM and F12 according to a volume ratio of 2:1;

E. inoculation of vaginal epithelial cells:

(2) Immersing culture with HVE-2 culture mediumRaising the temperature at 37 ℃ and 5% CO ₂ Culturing in an incubator for 4 days;

the HVE-2 culture solution is prepared according to the following method: adding 5% fetal bovine serum, 4.5 mu mol/mL glutamine, 22 mu g/mL adenine, 10 mu g/mL insulin, 1.5 mu g/mL hydrocortisone, 6ng/mL fibroblast growth factor, 12ng/mL transferrin, 1.5ng/mL EGF,0.2nmol/mL triiodothyronine, 8nmol/mL isoprenaline to a base solution of a culture medium mixed by DMEM and F12 according to a volume ratio of 2:1;

F. culture of vaginal mucosa model:

(2) The HVE-2 culture solution was replaced with HVE-3 culture solution at 37deg.C with 5% CO ₂ Culturing in an incubator for 10 days to obtain a double-layer vaginal mucosa model with a matrix layer containing fibroblasts and an epithelial layer containing vaginal epithelial cells;

the HVE-3 culture solution is prepared according to the following method: a medium in which DMEM and F12 were mixed at a volume ratio of 2:1 was used as a base solution, to which 5% fetal bovine serum, 3.0. Mu. Mol/mL glutamine, 18. Mu. G/mL adenine, 12. Mu. G/mL insulin, 1.5. Mu. G/mL hydrocortisone, 5.0ng/mL fibroblast growth factor, 15ng/mL transferrin, 3.5ng/mL EGF, 0.25. Mu. Mol/mL triiodothyronine, 8. Mu. Mol/mL isoprenaline, 60. Mu. G/mL vitamin C, 1.2. Mu. Mol/mL CaCl were added ₂ 。

Example 3

This embodiment differs from embodiment 1 in that:

the serum-free epithelial cell culture medium in the step A is based on a culture medium mixed by DMEM and F12 according to a volume ratio of 3:1, 6.0 mu mol/mL of glutamine, 5.0ng/mL of EGF,30 mu g/mL of BPE,15ng/mL of insulin, 2.0 mu g/mL of hydrocortisone and 0.4 mu mol/mL of CaCl are added into the culture medium ₂ 。

Example 4

A. vulvar epidermoid carcinoma cell line a431 cell culture:

resuspension of vulvar epidermoid carcinoma cell line A431 cells in DMEM medium containing 10% fetal bovine serum, 2.0. Mu. Mol/mL glutamine, 0.1. Mu. Mol/mL sodium pyruvate and 4.5g/L high sugar, 37℃and 5% CO ₂ The incubator is used for constructing an epithelial layer after subculturing until the seventh generation;

B. isolation and culture of vaginal fibroblasts:

C. preparation of a neutral collagen scaffold:

weighing collagen, placing the collagen into 0.1% acetic acid solution to prepare 10mg/mL collagen solution, placing the collagen solution on ice after complete dissolution, uniformly mixing the collagen solution and 10% fetal bovine serum DEME culture solution according to the volume ratio of 1:8, and adding 0.1M NaOH solution to adjust the pH to 7.4;

D. construction of a matrix layer containing fibroblasts:

(1) Vaginal fibroblasts of the 10 th generation were prepared at a density of 8×10 with DMEM containing 20% fetal bovine serum ⁶ Cell suspension per mL;

(2) Uniformly mixing a cell suspension and a neutral collagen scaffold according to a volume ratio of 1:9, inoculating into a Transwell chamber, culturing for 3 hours at 37 ℃, and then adding HVE-1 culture solution, 37 ℃ and 5% CO into the inside and outside of the Transwell chamber respectively ₂ Culturing in an incubator for 4 days to form a matrix layer in the vaginal mucosa model;

the HVE-1 culture solution is prepared according to the following method: adding 10% fetal bovine serum into a basic solution which is a culture medium mixed by DMEM and F12 according to a volume ratio of 3:1, adding 6.0 mu mol/mL glutamine, 30 mu g/mL adenine, 15 mu g/mL insulin, 2.0 mu g/mL hydrocortisone, and adding 100 mu g/mL vitamin C,10ng/mL fibroblast growth factor and 20ng/mL transferrin;

E. inoculation of vulvar epidermoid carcinoma cell line a431 cells:

(1) Sucking the culture medium on the surface of the substrate layer, and preparing the A431 cell line into 5.0X10 by using HVE-2 culture solution ⁶ Inoculating a cell suspension to a substrate layer in a Transwell chamber;

the HVE-2 culture solution is prepared according to the following method: adding 5% fetal bovine serum, 6.0 mu mol/mL glutamine, 30 mu g/mL adenine, 15 mu g/mL insulin, 2.0 mu g/mL hydrocortisone, 2.0ng/mL fibroblast growth factor, 20ng/mL transferrin, 2.0ng/mL EGF,0.4nmol/mL triiodothyronine, 10nmol/mL isoprenaline to a base solution of a culture medium mixed by DMEM and F12 in a volume ratio of 3:1;

F. culture of vaginal mucosa model:

(2) The HVE-2 culture solution was replaced with HVE-3 culture solution at 37deg.C with 5% CO ₂ Culturing in an incubator for 8-12 days to obtain a double-layer vaginal mucosa model with a matrix layer containing fibroblasts and an epithelial layer containing a vulva epidermoid cancer cell line A431;

the HVE-3 culture solution is prepared according to the following method: a medium in which DMEM and F12 were mixed at a volume ratio of 3:1 was used as a base solution, to which 5% fetal bovine serum, 6.0. Mu. Mol/mL of glutamine, 30. Mu. G/mL of adenine, 15. Mu. G/mL of insulin, 2.0. Mu. G/mL of hydrocortisone, 10ng/mL of fibroblast growth factor, 20ng/mL of transferrin, 5.0ng/mL of EGF,0.4nmol/mL of triiodothyronine, 10nmol/mL of isoprenaline, 100. Mu. G/mL of vitamin C, 1.5. Mu. Mol/mL of CaCl were added ₂ 。

Example 5

This embodiment differs from embodiment 4 in that:

step A: vulvar epidermoid carcinoma cell line a431 cell culture:

resuspension of vulvar epidermoid carcinoma cell line A431 cells in DMEM medium containing 10% fetal bovine serum, 4.5. Mu. Mol/mL glutamine, 0.5. Mu. Mol/mL sodium pyruvate and 4.5g/L high sugar, 37℃and 5% CO ₂ The incubator was used for construction of the epithelial layer after subculture to seventh generation.

Example 6

This embodiment differs from embodiment 4 in that:

step A: vulvar epidermoid carcinoma cell line a431 cell culture:

resuspension of vulvar epidermoid carcinoma cell line A431 cells in DMEM medium containing 10% fetal bovine serum, 6.0. Mu. Mol/mL glutamine, 1. Mu. Mol/mL sodium pyruvate and 4.5g/L high sugar, 37℃and 5% CO ₂ The incubator was used for construction of the epithelial layer after subculture to seventh generation.

As can be seen from fig. 1A and fig. 1B, the vaginal mucosa model constructed by using normal human vaginal epithelial cell culture has similar structural characteristics to normal vaginal tissue, the epithelial layer is mainly divided into three layers, the uppermost layer is a differentiated and mature stratum corneum, the middle layer is a transitional layer, the lowermost layer is a basal layer, and the basal layer is a collagen layer containing fibroblasts, which indicates that the 3D vaginal mucosa model constructed by the application can be normally differentiated in vitro, and is suitable for physiological research of human normal genital tract epithelium, drug toxicity safety evaluation and in vitro vaginal irritation experiment.

As can be seen from fig. 2A and fig. 2B, the epithelial layer of the vaginal mucosa model constructed by using the vulva epidermoid carcinoma cell line a431 cannot differentiate mature stratum corneum, but the model has a certain barrier function, and the model has the advantages of short construction period, wide sources of seed cells, and relatively stable performance, and can meet the basic requirements of in vitro vaginal irritation experiments.

The foregoing is merely illustrative of specific embodiments of the present application, and the scope of the present application is not limited thereto, but any changes or substitutions within the technical scope of the present application should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. An in vitro construction method of a human vaginal mucosa model is characterized by comprising the following steps:

A. construction of a matrix layer containing fibroblasts:

B. inoculation of cells:

C. culture of vaginal mucosa model:

sucking the HVE-2 culture solution on the surface of the culture, and lifting the cell culture chamber to an air-liquid interface for gas-liquid surface culture; changing the culture medium into HVE-3 culture solution, and culturing to obtain a double-layer vaginal mucosa model;

2. The method of claim 1, wherein the step of isolating and culturing vaginal fibroblasts in step a comprises the steps of:

3. The method of claim 1, wherein the step of isolating and culturing vaginal epithelial cells in step B is as follows:

4. The method of claim 1, wherein the step B comprises culturing the exocrine epidermoid carcinoma cell line a431 cells by: the re-suspended vulva epidermoid carcinoma cell line A431 cells are deposited in DMEM culture solution containing 10% fetal calf serum, 2.0-6.0 mu mol/mL glutamine, 0.1-1 mu mol/mL sodium pyruvate and 4.5g/L high sugar, and are used for constructing epithelial layer after subculture until the seventh generation.

5. The method of claim 1, wherein the method of preparing the neutral collagen scaffold in step a comprises the steps of: weighing collagen, placing the collagen into 0.1% acetic acid solution to prepare 4-10 mg/mL collagen solution, placing the collagen solution on ice after complete dissolution, uniformly mixing the collagen solution and 10% fetal bovine serum DEME culture solution according to the volume ratio of 1:8, and adding 0.1M NaOH solution to adjust the pH to 7.2-7.4.

6. The construction method according to claim 1, wherein the first culturing temperature in the step A is 37 ℃, the first culturing time is 2 to 3 hours, the second culturing temperature is 37 ℃, and the second culturing time is 2 to 4 days.

7. The method of claim 1, wherein the culturing temperature in step B is 37℃and the culturing time is 4 days.

8. The method according to claim 1, wherein the culturing temperature in step C is 37℃and the culturing time is 8 to 12 days.

9. The method of claim 2 or 3, wherein the PBS solution contains 100U/mL penicillin and 100U/mL streptomycin.

10. The method of claim 3, wherein the EDTA concentration is 0.2mg/mL.