Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration

A gas-liquid interface, three-dimensional culture technology, applied in biochemical equipment and methods, 3D culture, tissue culture and other directions, can solve the problems of wasting time and material resources, easily affected by the external environment, instability and other problems, saving time and material resources , Reasonable design, simple operation effect

Inactive Publication Date: 2017-05-17
TARIM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In the prior art, the hair follicle regeneration model and the scarless healing model of embryonic skin have not been successfully established in vitro; the current culture method simulates the physiological environment of the body, ...

Method used

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  • Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration
  • Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration
  • Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration

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Experimental program
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Effect test

Embodiment 1

[0061] (1) Fabrication of gas-liquid interface support:

[0062] Nuclepore polycarbonate membrane, made of high-quality polycarbonate film, each Nucle-pore polycarbonate membrane is round, with different specifications and different pore diameters, use 40 μL rat tail type I collagen mixed with the same amount of PBS, sterile Spread evenly on the inner wall of a disposable 90mm-diameter petri dish under certain conditions, pick up the Nuclepore polycarbonate membrane with sterile ophthalmic forceps, spread it on a petri dish coated with rat tail collagen, seal the petri dish with a parafilm, and irradiate with ultraviolet light After 30 minutes, the rat tail type I collagen and the Nuclepore polycarbonate membrane were naturally dried, and then placed in a 4°C refrigerator for later use.

[0063] (2) Preparation of culture medium:

[0064] In the experiment, low-sugar MEM was used as the basic culture medium, and fetal calf serum (FCS) was added to make a 5% FCS+MEM culture me...

Embodiment 2

[0070] Get pregnant mice whose gestational age is between 14-17d (E14-E17), kill them by neck dislocation, soak in 75% ethanol for 10 min, wash 3 times in sterile PBS, and put The uterus was taken out and washed 3 times in sterile PBS to remove residual blood and mucus on the surface. After the uterus was cut open, the embryos with fetal membranes were removed and washed 3 times in sterile PBS. Tear the fetal membrane with forceps and take out the fetal mouse. Rinse the fetal mouse in sterile PBS and cut it with ophthalmic scissors along the two sides of the back, carefully peel off the back skin of the fetal mouse with tweezers, and cut the embryonic skin into a regular quadrilateral with a micro-ophthalmological scalpel. And use a puncher to create an incision in the center of the quadrilateral skin, measure and record the diameter of the incision.

[0071] Add 2mL of the prepared culture solution into a 30mm petri dish, float the treated Nucle-pore polycarbonate membrane ...

Embodiment 3

[0073] On the Nuclepore polycarbonate membrane, add 2 μL of stem cell proliferation marker EdU dropwise, use sterile ophthalmic forceps to transfer the dissected outer root sheath of the hair follicle to the floating Nuclepore polycarbonate membrane, and remove the hair follicle outer root sheath and stem cell proliferation marker Fully exposed to EdU, placed at 37°C, 5% CO 2 Cultured in the incubator for 12 h, immunofluorescence staining was used to observe the proliferation state of hair follicle stem cells ( Figure 8 ).

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Abstract

The method provides a gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration. The method comprises the following steps: regenerating a hair follicle by culturing the outer root sheath of a vibrissa hair follicle, reestablishing the three-dimensional morphology of the hair follicle, simulating a body environment in a culture chamber, and culturing the outer root sheath of the hair follicle and embryo skin by utilizing the characteristics that the survival time of new hair follicles is relatively long, the neomorphosis is regular, and the hair follicles are not easy to collapse. Animal tissues (the hair follicles and fetal rat skin) are cultured on a gas-liquid interface by simulating the body environment, the cultured animal hair follicles and embryo skin have certain physiological functions, and the embryo skin can be healed without scars after being subjected to manual minimally invasive surgery; the gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration is a method for culturing stem cells and rapidly establishing a medical regeneration model, and hair follicle regeneration and skin scarless healing mechanisms can be explored outside the body; the design is reasonable, the operation is simple, and an in vitro tissue regeneration and wound healing model can be easily established in short time.

Description

[0001] Technical field: [0002] The invention relates to the technical field of skin and hair follicle regeneration, and specifically relates to a three-dimensional air-liquid interface culture method for skin and hair follicle regeneration. [0003] Background technique: [0004] There is skin around the hair follicle. As the largest organ of the body, the skin wraps the whole body. It is also the first important protective barrier between the internal environment and the natural environment. It protects the body from various factors in the environment and maintains the stability of the internal environment. state. Among them, the hair follicle, as the main appendage of the skin, has begun to develop during the embryonic period. The hair follicle is a sac-like tissue surrounded by the root of the hair. The mature hair follicle consists of an outer root sheath and an inner root sheath in the epithelial part and a dermal papilla and a dermal sheath in the dermal part. The dev...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2513/00
Inventor 李树伟王海涛周航震李娴
Owner TARIM UNIV
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