A method for constructing a human vaginal epithelial model in vitro

Abstract

An in vitro method for constructing a human vaginal epithelial model, which uses two stages of submerged culture and air-liquid surface culture to form a multilayered in vitro 3D model. During the submerged culture process, a low concentration of Ca 2+ (0.7~1.5mM) prevents the differentiation of vaginal epithelial cells; vitamin D is added to ensure that there are enough epithelial cells to maintain cell differentiation in the subsequent gas-liquid culture stage. In the gas-liquid interface culture stage, all-trans retinoic acid and 17 β-estradiol are added to the differentiation medium, among which all-trans retinoic acid can inhibit the continuous keratinization of vaginal epithelial cells and prevent excessive keratinization to form a stratum corneum. 17 β-estradiol enhances the barrier function of the vaginal epithelial model. Therefore, the joint use of all-trans retinoic acid and 17 β-estradiol can ensure that the epithelial layer of the vaginal epithelial model cannot differentiate into a mature stratum corneum, but still has a certain barrier function.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering, and more specifically relates to an in vitro construction method of a human vaginal epithelial model. Background Art

[0002] The human vaginal mucosa is composed of non-keratinized stratified squamous epithelium rich in glycogen and a basement membrane rich in fiber. Its structural characteristics make it a key site for external microbial invasion and a natural barrier for the human body to defend against viral and bacterial invasion. Frequent use of contraceptives, bactericides, female care products, and special therapeutic drugs can induce local irritation side effects on the mucosa, causing damage to the vaginal mucosal epithelium and making it extremely susceptible to infection with various pathogens. Therefore, detecting the irritation of substances in contact with human mucosa is the key to product quality and safety evaluation.

[0003] Traditional testing of cosmetic raw materials, product formulas, and final products is mainly completed through animal experiments. Due to the large individual differences in animal models and the lack of uniform evaluation standards, the results of animal experiments cannot accurately evaluate their safety and efficacy, and the results are poorly reproducible. Moreover, for products frequently used by women, animal models usually cannot distinguish the mildness of the products, nor can they compare the differences between formulas. In order to meet the development needs of cosmetics and other products, animal alternative testing has emerged to evaluate its feasibility in replacing traditional animal testing for safety evaluation. Therefore, it is of great significance to establish a research model that is close to the physiological state of the human reproductive tract epithelium. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide an in vitro method for constructing a human vaginal epithelial model.

[0005] Specifically, the present invention provides an in vitro method for constructing a human vaginal epithelial model, the method comprising the following steps:

[0006] Step 1: The specific method for preparing human vulvar epidermoid carcinoma cell line A431 cells is as follows:

[0007] A431 cells were taken, revived and expanded, and P42 cells were used for trypsin digestion to prepare a single cell suspension. The cell density was adjusted to 5.0×10 5 / mL~5.0×10 6 Pieces / mL;

[0008] Inoculate in a T75 culture flask, add cell culture medium 1, gently shake the culture flask to disperse the cells evenly, and place in an incubator at 37°C and 5% CO2 for culture.

[0009] The composition of A431 cell culture medium 1 was prepared as follows: DMEM medium containing 4.5 g / L glucose and 110 mg / L sodium pyruvate was used as the basal medium, and 10% fetal bovine serum and 1% gentamicin were added.

[0010] Step 2: The specific method of submerged inoculation culture of the human vaginal epithelium model is as follows:

[0011] Take the A431 cells in the logarithmic growth phase obtained in step 1, resuspend the A431 cells in cell culture medium 1 to a density of 2 million ± 500,000 / ml; take an appropriate amount of resuspended A431 cells and culture them at a density of 800,000 to 1.2 million / cm 2 The cells were inoculated at a high density in the Transwell chamber. 200ul of cell suspension was added to each Transwell chamber. The cell culture Transwell chamber was then placed in a 6-well plate. 1ml of cell culture medium 2 was added to each 6-well plate. The plates were placed at room temperature for 2h. 3ml of cell culture medium 2 was then added to the outside of the Transwell chamber to level the inside and outside interfaces of the Transwell chamber. The plates were then placed in an incubator at 37°C and 5% CO2 for 1 day. Cell culture medium 3 was then replaced and the plates were cultured for another 2 days to allow tight junctions to form between the cells inside the Transwell chamber.

[0012] Cell culture medium 2 was prepared as follows: DMEM as the basal medium, supplemented with 0.1-0.3% fetal bovine serum, 10-30ug / ml bovine pituitary extract BPE, 3-6μg / mL insulin, 0.05-0.2μg / mL hydrocortisone, 0.4-2mM L-glutamine, 1-1.5% gentamicin, 0.7-1.5mM calcium chloride, and 10-20mg vitamin D. -6 ~10 -9 mmol / L. The medium 2 is a low concentration of calcium, which can prevent the differentiation of vaginal epithelial cells; vitamin D is added, which can induce the proliferation of vaginal epithelial cells, enhance the adhesion between cells, and promote the maturation of vaginal epithelium. The addition of factors in the above culture medium simulates the in vivo microenvironment required for cell growth, so that cell growth or inhibition reaches a physiological balance, can better proliferate and differentiate, and ensure that there are enough epithelial cells to maintain cell differentiation in the subsequent gas-liquid culture stage.

[0013] Cell culture medium 3 was prepared as follows: DMEM as the basal medium, supplemented with 0.1-0.3% fetal bovine serum, 10-30ug / ml bovine pituitary extract BPE, 3-6μg / mL insulin, 0.1-0.4ng / ml EGF epidermal growth factor, 0.05-0.2μg / mL hydrocortisone, 0.4-2mM L-glutamine, 1-1.5% gentamicin, 0.7-1.5mM calcium chloride, and 10-20mg vitamin D.-6 ~10 -9 mmol / L.

[0014] Step 3: Immersion culture of human vaginal epithelial model:

[0015] The cell culture medium 3 inside and outside the Transwell chamber in step 2 was discarded, and the culture medium in the 6-well plate was replaced with differentiation culture medium 4 for immersion culture for 1 day.

[0016] Step 4: Air-liquid culture of human vaginal epithelial model:

[0017] The culture medium inside and outside the Transwell chamber in step 3 was discarded, and the Transwell chamber was placed on a sterile chamber stand. The culture medium outside the Transwell chamber was replaced with differentiation medium 4, so that the liquid surface and the upper surface of the cells in the Transwell chamber were at the same level to facilitate the formation of an air-liquid interface and start differentiation culture. No culture medium was added to the Transwell chamber. After replacing differentiation medium 4, the Transwell chamber was cultured at the air-liquid interface for 5 days, and differentiation medium 4 was replaced every 3 days to obtain a mature human vaginal epithelial model.

[0018] Differentiation medium 4 was prepared as follows: DMEM as basal medium, supplemented with 0.1~0.3% fetal bovine serum, 10~30ug / ml bovine pituitary extract BPE, 3~6μg / mL insulin, 0.1~0.4ng / ml EGF epidermal growth factor, 0.05~0.2μg / mL hydrocortisone, 0.4~2mM L-glutamine, 0.7~1.5mM calcium chloride, 1~1.5% gentamicin, and 10% vitamin D. -6 ~10 -9 mmol / L, all-trans retinoic acid 0.01~0.1umol / L, 17 β-estradiol 10~100nmol / L. In the gas-liquid interface culture stage, all-trans retinoic acid and 17 β-estradiol are added to the differentiation medium 4, wherein all-trans retinoic acid can inhibit the continuous keratinization of vaginal epithelial cells, prevent excessive keratinization to form a stratum corneum, and is conducive to the formation of a non-keratinized, stratified squamous epithelial structure, while also regulating immune function and enhancing the anti-inflammatory effect of vaginal epithelial cells; wherein the addition of 17 β-estradiol can increase the accumulation of glycogen in basal cells, and can also upregulate the expression of vaginal epithelial tight junction proteins, thereby enhancing the barrier function of the vaginal epithelial model. Therefore, the joint use of all-trans retinoic acid and 17 β-estradiol can ensure that the epithelial layer of the vaginal epithelial model cannot differentiate into a mature stratum corneum, but still has a certain barrier function.

[0019] The present invention has the following beneficial technical effects:

[0020] 1. The present invention discloses an in vitro method for constructing a human vaginal epithelial model, which uses human vulvar epidermoid carcinoma A431 cells as seed cells. The cell line is reliable and stable, and the constructed model has good reproducibility. Moreover, while ensuring the formation of the model's multi-layered structure, the gas-liquid surface culture time of the model construction is shortened, thereby reducing production costs.

[0021] 2. The basic components of the culture medium used in the present invention can meet the basic physiological needs of vaginal epithelial cells; during the submerged culture process, low concentrations of Ca 2+ (0.7~1.5mM) prevents the differentiation of vaginal epithelial cells; adding vitamin D can reduce the vaginal pH, promote the proliferation of vaginal epithelial cells, enhance the adhesion between cells, and promote the maturation of vaginal epithelium. The addition of factors in the above culture medium simulates the in vivo microenvironment required for cell growth, so that cell growth or inhibition reaches a physiological balance, can better proliferate and differentiate, and ensure that there are enough epithelial cells to maintain cell differentiation in the subsequent gas-liquid culture stage. In the gas-liquid interface culture stage, all-trans retinoic acid and 17β-estradiol are added to the culture medium, among which all-trans retinoic acid can inhibit the continuous keratinization of vaginal epithelial cells, prevent excessive keratinization to form a stratified squamous epithelial structure, and at the same time regulate immune function and enhance the anti-inflammatory effect of vaginal epithelial cells; among which, the addition of 17β-estradiol can increase the accumulation of glycogen in basal cells and upregulate the expression of tight junction proteins in vaginal epithelium, thereby enhancing the barrier function of the vaginal epithelial model. Therefore, the combined use of all-trans retinoic acid and 17β-estradiol can ensure that the epithelial layer of the vaginal epithelial model cannot differentiate into a mature stratum corneum, but still has a certain barrier function.

[0022] 3. The three-dimensional human vaginal epithelial model obtained by the present invention replicates the morphology and characteristics of the vaginal epithelium and better simulates the in vivo environment and normal physiological state of the body. The model can be used to conduct vaginal irritation experiments on female care products, which is closer to the real situation in the body and the experimental results are more accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the histological H&E staining picture of the human vaginal epithelial model provided in Example 1 of the present application;

[0024] Figure 2 This is the histological H&E staining picture of the human vaginal epithelial model provided in Example 2 of the present application;

[0025] Figure 3 This is the histological H&E staining picture of the human vaginal epithelial model provided in Example 3 of the present application;

[0026] Figure 4The histological H&E staining picture of the human vaginal epithelial model provided in Comparative Example 1 of the present application;

[0027] Figure 5 The histological H&E staining picture of the human vaginal epithelial model provided in Comparative Example 2 of this application;

[0028] Figure 6 This is the histological H&E staining picture of the human vaginal epithelial model provided in Comparative Example 3 of the present application. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further explained and illustrated by specific embodiments and drawings. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0030] Example 1

[0031] Example 1 of the present application provides a method for constructing a human vaginal epithelial model, the method comprising:

[0032] Step 1: The specific method for preparing human vulvar epidermoid carcinoma cell line A431 cells is as follows:

[0033] A431 cells were taken, revived and expanded, and P42 cells were used for trypsin digestion to prepare a single cell suspension. The cell density was adjusted to 5.0×10 5 The cells were inoculated into a T75 culture flask, and cell culture medium 1 was added. The culture flask was gently shaken to disperse the cells evenly, and then cultured in an incubator at 37°C and 5% CO2.

[0034] The composition of A431 cell culture medium 1 was prepared as follows: DMEM medium containing 4.5 g / L glucose and 110 mg / L sodium pyruvate was used as the basal medium, and 10% fetal bovine serum and 1% gentamicin were added.

[0035] Step 2: The specific method of submerged inoculation culture of the human vaginal epithelium model is as follows:

[0036] Take the A431 cells in the logarithmic growth phase obtained in step 1, resuspend the A431 cells in cell culture medium 1 to a density of 2 million / ml; take an appropriate amount of resuspended A431 cells and culture them at a density of 800,000 / cm 2Inoculation density: Inoculate in the Transwell chamber, add 200ul cell suspension to each Transwell chamber, then place the cell culture Transwell chamber in a 6-well plate, add 1ml cell culture medium 2 to each 6-well plate, place at room temperature for 2h, then add 3ml cell culture medium 2 to the outside of the Transwell chamber, make the inside and outside interfaces of the Transwell chamber level, place in an incubator at 37℃ and 5% CO2 for 1 day; then replace cell culture medium 3 and continue to culture for 2 days. This allows tight junctions to form between cells inside the Transwell chamber.

[0037] Cell culture medium 2 was prepared as follows: DMEM as the basal medium, supplemented with 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1% gentamicin, 1.0mM calcium chloride, and 10mg vitamin D. -6 mmol / L.

[0038] Cell culture medium 3 was prepared as follows: DMEM as the basal medium, supplemented with 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2ng / ml EGF epidermal growth factor, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1% gentamicin, 1.0mM calcium chloride, and 10mg vitamin D. -6 mmol / L.

[0039] Step 3: Immersion culture of human vaginal epithelial model:

[0040] The cell culture medium 3 inside and outside the Transwell chamber in step 2 was discarded, and the culture medium in the 6-well plate was replaced with differentiation culture medium 4 for immersion culture for 1 day.

[0041] Step 4: Air-liquid culture of human vaginal epithelial model:

[0042] The culture medium inside and outside the Transwell chamber in step 3 was discarded, and the Transwell chamber was placed on a sterile chamber stand. The culture medium outside the Transwell chamber was replaced with differentiation medium 4, so that the liquid surface and the upper surface of the cells in the Transwell chamber were at the same level to facilitate the formation of an air-liquid interface and start differentiation culture. No culture medium was added to the Transwell chamber. After replacing differentiation medium 4, the Transwell chamber was cultured at the air-liquid interface for 5 days, and differentiation medium 4 was replaced every 3 days to obtain a mature human vaginal epithelial model.

[0043] Differentiation medium 4 was prepared as follows: DMEM as the basal medium, supplemented with 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2ng / ml EGF epidermal growth factor, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1.0mM calcium chloride, 1% gentamicin, and 10% vitamin D. -6 mmol / L, all-trans retinoic acid 0.1umol / L, 17 β-estradiol 100nmol / L.

[0044] Example 2

[0045] Example 2 of the present application provides a method for constructing a human vaginal epithelial model, the method comprising:

[0046] Step 1: The specific method for preparing human vulvar epidermoid carcinoma cell line A431 cells is as follows:

[0047] A431 cells were taken, revived and expanded, and P42 cells were used for trypsin digestion to prepare a single cell suspension. The cell density was adjusted to 5.0×10 6 The cells were inoculated into a T75 culture flask, and cell culture medium 1 was added. The culture flask was gently shaken to disperse the cells evenly, and then cultured in an incubator at 37°C and 5% CO2.

[0048] The composition of A431 cell culture medium 1 was prepared as follows: DMEM medium containing 4.5 g / L glucose and 110 mg / L sodium pyruvate was used as the basal medium, and 10% fetal bovine serum and 1% gentamicin were added.

[0049] Step 2: The specific method of submerged inoculation culture of the human vaginal epithelium model is as follows:

[0050] Take the A431 cells in the logarithmic growth phase obtained in step 1, resuspend the A431 cells in cell culture medium 1 to a density of 2 million / ml; take an appropriate amount of resuspended A431 cells and culture them at a density of 1 million / cm 2 The cell suspension was inoculated into the Transwell chamber at a density of 200 μl. Then, the cell culture Transwell chamber was placed in a 6-well plate. 1 ml of cell culture medium 2 was added to each 6-well plate. The plate was placed at room temperature for 2 h. Then, 3 ml of cell culture medium 2 was added to the Transwell chamber to make the inner and outer interfaces of the Transwell chamber level. The plate was placed at 37°C and 5% CO. The cells were cultured in a culture incubator with the same conditions for 1 day; then the cell culture medium 3 was replaced and cultured for another 2 days to form tight junctions between the cells in the Transwell chamber.

[0051] Cell culture medium 2 was prepared as follows: DMEM as the basal medium, supplemented with 0.1% fetal bovine serum, 15ug / ml bovine pituitary extract BPE, 3μg / mL insulin, 0.1μg / mL hydrocortisone, 0.4mM L-glutamine, 1% gentamicin, 0.8mM calcium chloride, and 10% vitamin D. -7 mmol / L.

[0052] Cell culture medium 3 was prepared as follows: DMEM was used as the basal medium, supplemented with 0.1% fetal bovine serum, 15ug / ml bovine pituitary extract BPE, 3μg / mL insulin, 0.1ng / ml EGF epidermal growth factor, 0.1μg / mL hydrocortisone, 0.4mM L-glutamine, 1% gentamicin, 0.8mM calcium chloride, and 10mg vitamin D. -7 mmol / L.

[0053] Step 3: Immersion culture of human vaginal epithelial model:

[0054] The cell culture medium 3 inside and outside the Transwell chamber in step 2 was discarded, and the culture medium in the 6-well plate was replaced with differentiation culture medium 4 for immersion culture for 1 day.

[0055] Step 4: Air-liquid culture of human vaginal epithelial model:

[0056] The culture medium inside and outside the Transwell chamber in step 3 was discarded, and the Transwell chamber was placed on a sterile chamber stand. The culture medium outside the Transwell chamber was replaced with differentiation medium 4, so that the liquid surface and the upper surface of the cells in the Transwell chamber were at the same level to facilitate the formation of an air-liquid interface and start differentiation culture. No culture medium was added to the Transwell chamber. After replacing differentiation medium 4, the Transwell chamber was cultured at the air-liquid interface for 5 days, and differentiation medium 4 was replaced every 3 days to obtain a mature human vaginal epithelial model.

[0057] Differentiation medium 4 was prepared as follows: DMEM as the basal medium, supplemented with 0.1% fetal bovine serum, 15ug / ml bovine pituitary extract BPE, 3μg / mL insulin, 0.1ng / ml EGF epidermal growth factor, 0.1μg / mL hydrocortisone, 0.4mM L-glutamine, 1% gentamicin, 0.8mM calcium chloride, and 10mg vitamin D. -7 mmol / L, all-trans retinoic acid 0.05umol / L, 17 β-estradiol 50nmol / L.

[0058] Example 3

[0059] Example 3 of the present application provides a method for constructing a human vaginal epithelial model, the method comprising:

[0060] Step 1: The specific method for preparing human vulvar epidermoid carcinoma cell line A431 cells is as follows:

[0061] A431 cells were taken, revived and expanded, and P42 cells were used for trypsin digestion to prepare a single cell suspension. The cell density was adjusted to 5.0×10 5 The cells were inoculated into a T75 culture flask, and cell culture medium 1 was added. The culture flask was gently shaken to disperse the cells evenly, and then cultured in an incubator at 37°C and 5% CO2.

[0062] The composition of A431 cell culture medium 1 was prepared as follows: DMEM medium containing 4.5 g / L glucose and 110 mg / L sodium pyruvate was used as the basal medium, and 10% fetal bovine serum and 1% gentamicin were added.

[0063] Step 2: The specific method of submerged inoculation culture of the human vaginal epithelium model is as follows:

[0064] Take the A431 cells in the logarithmic growth phase obtained in step 1, resuspend the A431 cells in cell culture medium 1 to a density of 2.5 million / ml; take an appropriate amount of resuspended A431 cells and resuspend them at a density of 800,000 / cm 2 Inoculation density: Inoculate in Transwell chambers, add 200ul cell suspension into each Transwell chamber, then place the cell culture Transwell chambers into 6-well plates, add 1ml cell culture medium 2 into each 6-well plate, place at room temperature for 2h, then add 3ml cell culture medium 2 into the Transwell chambers, make the interface between the inside and outside of the Transwell chambers level, place in an incubator at 37℃ and 5% CO2 for 1 day, then replace cell culture medium 3 and continue to culture for 2 days. This allows tight junctions to form between cells inside the Transwell chambers.

[0065] Cell culture medium 2 was prepared as follows: DMEM as the basal medium, supplemented with 0.3% fetal bovine serum, 10ug / ml bovine pituitary extract BPE, 6μg / mL insulin, 0.1μg / mL hydrocortisone, 2mM L-glutamine, 1% gentamicin, 1.5mM calcium chloride, and 10 -9 mmol / L.

[0066] Cell culture medium 3 was prepared as follows: DMEM as the basal medium, supplemented with 0.3% fetal bovine serum, 10ug / ml bovine pituitary extract BPE, 6μg / mL insulin, 0.4ng / ml EGF epidermal growth factor, 0.1μg / mL hydrocortisone, 2mM L-glutamine, 1% gentamicin, 1.5mM calcium chloride, and 10 -9 mmol / L.

[0067] Step 3: Immersion culture of human vaginal epithelial model:

[0068] The cell culture medium 3 inside and outside the Transwell chamber in step 2 was discarded, and the culture medium in the 6-well plate was replaced with differentiation culture medium 4 for immersion culture for 1 day.

[0069] Step 4: Air-liquid culture of human vaginal epithelial model:

[0070] The culture medium inside and outside the Transwell chamber in step 3 was discarded, and the Transwell chamber was placed on a sterile chamber stand. The culture medium outside the Transwell chamber was replaced with differentiation medium 4, so that the liquid surface and the upper surface of the cells in the Transwell chamber were at the same level to facilitate the formation of an air-liquid interface and start differentiation culture. No culture medium was added to the Transwell chamber. After replacing differentiation medium 4, the Transwell chamber was cultured at the air-liquid interface for 5 days, and differentiation medium 4 was replaced every 3 days to obtain a mature human vaginal epithelial model.

[0071] Differentiation medium 4 was prepared as follows: DMEM as the basal medium, supplemented with 0.3% fetal bovine serum, 10ug / ml bovine pituitary extract BPE, 6μg / mL insulin, 0.4ng / ml EGF epidermal growth factor, 0.1μg / mL hydrocortisone, 2mM L-glutamine, 1.5mM calcium chloride, 1% gentamicin, and 10mg vitamin D. -9 mmol / L, all-trans retinoic acid 0.01umol / L, 17 β-estradiol 30nmol / L.

[0072] Comparative Example 1

[0073] The present comparative example 1 provides a method for constructing a human vaginal epithelial model, which differs from the method of Example 1 in that: the differentiation medium 4 is prepared according to the following method: DMEM is used as the basic medium, and 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2ng / ml EGF epidermal growth factor, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1.0mM calcium chloride, 1% gentamicin, and 10% vitamin D are added. -6mmol / L, 17β-estradiol 100nmol / L. The specific culture method is the same as that in Example 1.

[0074] Comparative Example 2

[0075] Comparative Example 2 provides a method for constructing a human vaginal epithelial model, which differs from Example 1 in that: differentiation medium 4 is prepared according to the following method: DMEM is used as the basic medium, and 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2ng / ml EGF epidermal growth factor, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1.0mM calcium chloride, 1% gentamicin, and 10% vitamin D are added. -6 mmol / L, all-trans retinoic acid 0.1umol / L. The specific culture method is the same as in Example 1.

[0076] Comparative Example 3

[0077] Comparative Example 3 provides a method for constructing a human vaginal epithelial model. The difference between the construction method and Example 1 is that the cell culture medium 2 is prepared according to the following method: DMEM is used as the basic culture medium, and 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1% gentamicin, 1.8mM calcium chloride, and 10% vitamin D are added. -6 mmol / L. The specific culture method is the same as in Example 1.

[0078] Test example

[0079] The histological structure, tissue viability, and barrier function of the human vaginal epithelial models prepared in Examples 1-3 and Comparative Examples 1-3 of the present application were tested, as described in detail below.

[0080] 1. Histological structure detection of human vaginal epithelium model

[0081] The human vaginal epithelial models provided in Examples 1 to 3 and Comparative Examples 1 to 3 of the present application were sent to a third-party testing company to test the histological structure of the models, and the results were obtained. Figure 1 to Figure 6 The histological structure of the human vaginal epithelial model obtained by the construction method of Example 1 is as follows Figure 1 As shown in the figure, the basal layer of the model has many cells, the cells are closely arranged, and the epithelial layer has not differentiated into a mature stratum corneum, but it still has a certain barrier function and can meet the basic requirements of in vitro vaginal irritation experiments. The histological structure of the human vaginal epithelial model obtained by the construction method of Example 2 is shown in the figure. Figure 2 As shown in Figure 3, the basal layer of the model has few cells and more vacuoles. The histological structure of the human vaginal epithelial model obtained by the construction method of Example 3 is as follows: Figure 3 As shown in FIG. 1 , the basal layer of the model has few cells and more vacuoles. The histological structure of the human vaginal epithelial model obtained by the construction method of Comparative Example 1 is shown in FIG. Figure 4 As shown in FIG. 1 , it is obvious that the epithelial layer of the model differentiates into a mature stratum corneum. The histological structure of the human vaginal epithelial model obtained by the construction method of Comparative Example 2 is shown in FIG. Figure 5 As shown, the cells in the model cell layer are loosely arranged, have many cell vacuoles, and have poor barrier function.

[0082] The histological structure of the human vaginal epithelial model obtained by the construction method of Comparative Example 3 is as follows Figure 6 As shown, there are few cells in the basal layer of this model, many cell vacuoles, and poor barrier function.

[0083] 2. Detection of tissue viability in human vaginal epithelial model

[0084] The tissue viability of the human vaginal epithelial models provided in Examples 1 to 3 and the models provided in Comparative Examples 1 to 3 were tested under the same conditions.

[0085] The specific steps of tissue viability detection include: adding 0.3mL of MTT (thiazolyl blue) with a concentration of 1mg / mL to each well of a 24-well plate. Gently wipe the residual liquid inside and outside the model with a sterile cotton swab. Transfer the model to a 24-well plate so that it is fully in contact with the MTT solution. Place the 24-well plate in a CO2 incubator at 37±1℃, 5% CO2, and 95% relative humidity for 3h±5min. After the incubation, discard the MTT solution and wash the outer surface of the model with PBS solution. Use a sterile cotton swab to wipe the outer surface of the model dry and transfer it to a new 24-well plate. Add 1.5mL of isopropanol to the inside and outside of the model, and seal the 24-well plate with a sealing film to prevent the isopropanol from volatilizing. Leave it at 4℃ overnight or shake it on a plate shaker for 2h. Use a pipette tip to pierce the model so that the isopropanol extract flows out of the model into the 24-well plate and mix the isopropanol extract. 3 portions of 200 µL isopropanol extract were taken from each well and added to a 96-well plate, with isopropanol used as a blank control. The absorbance OD value of each group was measured using an ELISA reader at a wavelength of 570 nm, and the OD570 value was the tissue viability of the human vaginal epithelial model. The test data are shown in Table 1.

[0086] Table 1: Tissue viability of the human vaginal epithelium model

[0087]

[0088] Taking the tissue viability of Example 1 as 100%, it can be seen from the data in Table 1 that the human vaginal epithelial model prepared in Example 1 has good tissue viability.

[0089] 3. Barrier function testing of human vaginal epithelial model

[0090] The human vaginal epithelial models provided in Examples 1 to 3 and the models provided in Comparative Examples 1 to 3 were tested for ET50 under the same conditions.

[0091] (1) Transfer the human vaginal epithelial model to a sterile 6-well plate and add 0.9 mL of differentiation medium 4 to each well. Use a pipette to draw 80 μL of PBS solution and slowly drip it onto the surface of the model as the negative control group. Draw 80 μL of 1.0% Triton X-100 and slowly drip it onto the surface of the model to ensure that the reagent can cover the surface of the model as much as possible as the experimental group. After the model is administered, all 6-well plates are transferred to a constant temperature incubator (37±1℃, 5±1% CO2, 95% relative humidity). The administration time is 0h, 0.5h, 1h, and 2h, respectively.

[0092] (2) After the incubation time is over, wash the model with 10 mL of PBS solution to remove residual Triton X-100. Then use a sterile cotton swab to wipe the inner and outer surfaces of the model dry. Transfer the cleaned and dried model to a 24-well plate with 1 mg / mL MTT test solution, add 300 ul of 1 mg / mL MTT test solution to each well, and place the 24-well plate in a constant temperature incubator (37±1℃, 5±1% CO2, 95% relative humidity) for 3 h.

[0093] (3) After the MTT incubation is completed, gently remove the MTT solution from the well plate and add PBS solution to the well plate to wash twice. After washing, wipe the bottom of the model with absorbent paper and transfer it to a new 24-well plate. Add 1.5 mL of isopropanol inside and outside the model, which can dissolve the crystals produced by MTT. Use sealing film to seal the gap between the 24-well plates to prevent the evaporation of isopropanol from affecting the final volume. Let it stand at 4°C overnight to dissolve, or shake it on a well plate shaker for 2 hours.

[0094] (4) After dissolution is complete, use a pipette tip to pierce the model so that the isopropanol extract flows out of the model into the 24-well plate, and mix the isopropanol extract. For each model, pipette 3 portions of 200 μL of purple formazan solution into the same 96-well plate. Use isopropanol as a blank control. Use an ELISA reader to measure the absorbance OD value at a wavelength of 570 nm. The survival rate of each group at 0 h of stimulation is 100%, and the absorbance value is used as the detection index to calculate the tissue viability of the human vaginal epithelial model according to the following formula.

[0095] (5) Tissue viability at each time point in each group = (OD value at each time point in each group - OD value of blank control) / (OD value at 0 h in each group - OD value of blank control) * 100%.

[0096] (6) Within the same experimental group, the data of two time points when the tissue activity is about 50% are taken for plotting, and the linear regression equation is calculated. The time when the tissue activity is 50% is calculated according to the regression equation, which is the barrier index (ET50). The ET50 value represents the ability of the skin to tolerate exogenous irritants. The larger the ET50 value, the better the barrier function. The test data are shown in Table 2.

[0097] Table 2: Barrier function of human vaginal epithelium model

[0098]

[0099] It can be seen from the data in Table 2 that the ET50 value of Comparative Example 1 is higher than that of Example 1 because the human vaginal epithelial model tissue structure constructed in Comparative Example 1 differentiates a mature stratum corneum and can withstand the rapid penetration of exogenous irritants, but the tissue structure is inconsistent with the natural human vaginal epithelial structure.

[0100] Therefore, the human vaginal epithelial model prepared in Example 1 has a good barrier function.

[0101] In summary, it can be seen from the data in Tables 1 and 2 that the human vaginal epithelial model provided in Example 1 of the present application has good barrier function and high tissue viability.

[0102] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a human vaginal epithelial model in vitro, characterized in that: The following steps are involved: Step 1: Preparation of human vulvar epidermoid carcinoma cell line A431 cells. The specific method is as follows: A431 cells were taken, revived and expanded, and P42 cells were used for trypsin digestion to prepare a single cell suspension. The cell density was adjusted to 5.0×10 5 / mL~5.0×10 6 Pieces / mL; Inoculate in a T75 culture flask, add cell culture medium 1, shake the culture flask to disperse the cells evenly, and place in an incubator at 37°C and 5% CO2 for culture; Step 2: Submerged inoculation culture of human vaginal epithelial model, the specific method is as follows: Take the A431 cells in the logarithmic growth phase obtained in step 1, resuspend the A431 cells in cell culture medium 1 to a density of 2 million ± 500,000 / ml; take the resuspended A431 cells and culture them at a density of 800,000 to 1.2 million / cm 2 Inoculation density: Inoculate in Transwell chambers, add 200ul of cell suspension into each Transwell chamber, then place the cell culture Transwell chambers into 6-well plates, add 1ml of cell culture medium 2 into each 6-well plate, and place at room temperature for 2h, then add 3ml of cell culture medium 2 into the Transwell chambers to make the inner and outer interfaces of the Transwell chambers level, and place in an incubator at 37°C and 5% CO2 for 1 day; then replace cell culture medium 3, and continue culturing for 2 days to form tight junctions between cells inside the Transwell chambers; Step 3: Immersion culture of human vaginal epithelial model: The cell culture medium 3 inside and outside the Transwell chamber in step 2 was discarded, and the culture medium in the 6-well plate was replaced with differentiation medium 4 for immersion culture for 1 day; Step 4: Air-liquid culture of human vaginal epithelial model: The culture medium inside and outside the Transwell chamber in step 3 was discarded, and the Transwell chamber was placed on a sterile chamber stand. The culture medium outside the Transwell chamber was replaced with differentiation medium 4, so that the liquid surface and the upper surface of the cells in the Transwell chamber were at the same level, and differentiation culture was started; no culture medium was added to the Transwell chamber. After the differentiation medium 4 was replaced, the Transwell chamber was subjected to air-liquid interface culture for 5 days, and the differentiation medium 4 was replaced every 3 days to obtain a mature human vaginal epithelial model; The composition of the medium 2 in step 2 was prepared as follows: DMEM as the basal medium, supplemented with 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1% gentamicin, 1.0mM calcium chloride, and 10 -6 mmol / L; The composition of the cell culture medium 3 in step 2 was prepared according to the following method: DMEM as the basic medium, supplemented with 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2ng / ml EGF epidermal growth factor, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1% gentamicin, 1.0mM calcium chloride, and 10 -6 mmol / L; The composition of differentiation medium 4 in step 4 was prepared as follows: DMEM as the basal medium, supplemented with 0.2% fetal bovine serum, 25ug / ml bovine pituitary extract BPE, 5μg / mL insulin, 0.2ng / ml EGF epidermal growth factor, 0.2μg / mL hydrocortisone, 0.6mM L-glutamine, 1.0mM calcium chloride, 1% gentamicin, and 10% vitamin D. -6 mmol / L, all-trans retinoic acid 0.1umol / L, 17 β-estradiol 100nmol / L.

2. The in vitro construction method of the human vaginal epithelium model according to claim 1, characterized in that: The composition of the cell culture medium 1 in step 1 is prepared according to the following method: DMEM medium containing 4.5 g / L glucose and 110 mg / L sodium pyruvate is used as the basic medium, and 10% fetal bovine serum and 1% gentamicin are added.

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