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Preparation method of culture medium for constructing human vaginal mucosa model

A vaginal mucosa and preparation method technology, applied in the field of biomedicine, can solve problems such as limited sources of vaginal tissue, high requirements for culture conditions, and long model building time, achieving accurate and reliable experimental results, reducing production costs, and shortening the construction time.

Inactive Publication Date: 2021-12-31
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The RHVE model of the French SkinEthic company uses the vulvar epidermoid cancer cell line A431 as the seed cell. The cell line is stable and reliable, and can solve the problem of seed cell source. However, it only contains the epithelial layer composed of A431 cells and lacks stromal layer cells and matrix, so In terms of tissue structure, there is still a big difference with natural vaginal tissue
The EpiVaginaL vaginal model of MatTek in the United States and the human vaginal epithelium 3D model disclosed in the Chinese patent CN201710048227.7 have a highly similar tissue structure to the natural vaginal mucosa. Most of the cells are differentiated mature cells with low proliferative ability, and only a small number of basal cells have strong proliferative ability. Therefore, the requirements for large-scale expansion and culture in vitro are high, and mass production in vitro cannot be achieved to meet market demand.
The 3D model of human vaginal epithelium disclosed in Chinese patent CN201710048227.7 still has some defects, such as a single culture system that cannot adapt to the nutritional needs of cells at different stages, resulting in poor stratification of the model, weak barrier function, and the model takes a long time to build , it takes up to one month to construct a batch of models in vitro, and a too long production cycle will increase the uncontrollable factors in the process and affect the stability of the model. There is no commercial model of human vaginal mucosa

Method used

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  • Preparation method of culture medium for constructing human vaginal mucosa model
  • Preparation method of culture medium for constructing human vaginal mucosa model
  • Preparation method of culture medium for constructing human vaginal mucosa model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] An in vitro construction method of a human vaginal mucosa model, comprising the steps of:

[0048] A. Isolation and culture of vaginal epithelial cells:

[0049] (1) Place the human vaginal tissue in a petri dish, wash it 6 times with 4°C pre-cooled PBS solution, remove the submucosal tissue, cut the tissue block into a size of 0.2cm×0.2cm, add 1.5U / mL of dispersing Digest overnight at 4°C in enzymes;

[0050] (2) The epithelial layer and lamina propria of the vaginal mucosa were separated by ophthalmic forceps, and the epithelial layer of the vaginal mucosa was put into 0.25% trypsin-EDTA, digested at 37°C for 30 minutes, and the concentration of EDTA was 0.2 mg / mL;

[0051] (3) Use DMEM medium containing 10% fetal bovine serum to stop the digestion, filter through a 200-mesh sieve, centrifuge to remove the supernatant, and collect vaginal mucosal epithelial cells;

[0052] (4) Wash vaginal epithelial cells with PBS, resuspend the cell pellet in serum-free epithelial...

Embodiment 2

[0072] An in vitro construction method of a human vaginal mucosa model, comprising the steps of:

[0073] A. Isolation and culture of vaginal epithelial cells:

[0074] (1) Place the human vaginal tissue in a petri dish, wash it 6 times with 4°C pre-cooled PBS solution, remove the submucosal tissue, cut the tissue block into a size of 0.2cm×0.2cm, add 1.5U / mL of dispersing Digest overnight at 4°C in enzymes;

[0075] (2) The vaginal epithelial layer and lamina propria were separated with ophthalmic forceps, and the vaginal epithelial layer was placed in 0.25% trypsin-EDTA, digested at 37°C for 45 minutes, and the concentration of EDTA was 0.2 mg / mL;

[0076] (3) Use DMEM medium containing 10% fetal bovine serum to stop the digestion, filter through a 200-mesh sieve, centrifuge to remove the supernatant, and collect vaginal mucosal epithelial cells;

[0077] (4) Wash vaginal epithelial cells with PBS, resuspend the cell pellet in serum-free epithelial cell culture medium, obt...

Embodiment 3

[0097] The difference between this embodiment and embodiment 1 is:

[0098] The serum-free epithelial cell culture medium described in step A is based on the medium mixed with DMEM and F12 at a volume ratio of 3:1, to which 6.0 μmol / mL glutamine, 5.0 ng / mL EGF, and 30 μg / mL are added BPE, 15ng / mL insulin, 2.0μg / mL hydrocortisone, 0.4μmol / mL CaCl 2 .

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Abstract

The invention provides a preparation method of a culture medium for constructing a human vaginal mucosa model. The preparation method comprises the following steps: taking a culture medium formed by mixing DMEM and F12 according to a volume ratio of (1-3): 1 as a basic solution, and adding 5% fetal calf serum, 2.0-6.0 micromole / mL glutamine, 15-30 microgram / mL adenine, 5-15 microgram / mL insulin, 0.5-2.0 microgram / mL hydrocortisone, 1-10 ng / mL fibroblast growth factors, 5-20 ng / mL transferrin and 0.5-2.0 ng / mL epidermal growth factors, 0.1-0.4 nmol / mL triiodothyronine and 5 to 10 nmol / mL isoprenaline into the basic solution.

Description

[0001] This divisional application is based on the divisional application of the Chinese patent application with the application number 202110356820.4, the application date is April 1, 2021, and the invention title is "a method for constructing an in vitro reconstructed human vaginal mucosa model". technical field [0002] The invention belongs to the field of biomedicine, in particular to a preparation method for constructing a human vaginal mucosa model culture medium. Background technique [0003] The human vaginal mucosa consists of thick, nonkeratinized, stratified squamous epithelium rich in glycogen, with small numbers of other cell types such as macrophages and Langerhans cells. Underlying the epithelium is the lamina propria, which contains many elastic fibers and a dense vascular network, making the vaginal mucosa an excellent route for drug delivery for local and systemic treatments. However, due to long-term exposure to various pathogens in the lumen of the repro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0682C12N5/0693C12N2509/00C12N2500/32C12N2500/84C12N2501/11C12N2501/33C12N2501/30C12N2500/14C12N2500/40C12N2500/24C12N2501/10Y02A50/30
Inventor 李润芝卢永波张勇杰
Owner GUANGDONG BOXI BIO TECH CO LTD