Method for extracting and culturing amniotic stem cells
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A stem cell and amniotic membrane technology, applied in the field of culture and extraction of amniotic membrane stem cells, can solve the problems of low efficiency and low purity of stem cells, and achieve the effect of high extraction efficiency and high purity
Active Publication Date: 2019-09-10
AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV
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[0004] The purpose of the present invention is to provide a method for extracting and culturing amniotic membrane stem cells, aiming to solve the ...
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Embodiment 1
[0027] 1. Amniotic membrane preparation
[0028] Take normal cesarean section placenta without hepatitis, syphilis, HIV and other infectious diseases.
[0029] The amniotic membrane is mechanically separated from the placenta, and then the removed amniotic membrane is placed in a PBS container containing double antibodies and washed repeatedly to remove blood and debris.
[0030] 2. Extraction of amniotic membrane stem cells
[0031] 2.1. Shake the amniotic membrane with mixed solution 1 at room temperature and soak for 0.5-1 hour, then wash it with tris buffer solution; mixed solution 1 contains 0.03% (w / v) sodium lauryl sulfate and 0.1 %(w / v)
[0032] EDTA in Tris buffer;
[0033] 2.2. Gently scrape the surface of the amniotic membrane with a cell scraper, rinse with PBS for 3 to 5 times, and mechanically cut the amniotic membrane to a size of 2*2mm;
[0034] 2.3. Add the amniotic membrane fragments to the mixture 2, and then put it in 37℃, CO 2 The volume fraction of t...
Embodiment 2
[0042] Embodiment 2 is basically the same as Embodiment 1, the difference is:
[0043] Mixed solution 1 is tris buffer solution containing 0.05% (w / v) sodium lauryl sulfate and 0.5% (w / v) ethylenediaminetetraacetic acid;
[0044] The mixed solution 2 is a mixed solution of α-MEM medium containing 3 g / L type II collagenase, 0.1 g / L DNase I and 15% fetal bovine serum.
Embodiment 3
[0046] Embodiment 2 is basically the same as Embodiment 1, the difference is:
[0047] Mixed solution 1 is tris buffer solution containing 0.04% (w / v) sodium lauryl sulfate and 0.3% (w / v) ethylenediaminetetraacetic acid;
[0048] The mixed solution 2 is a mixed solution of α-MEM medium containing 2.5g / L type II collagenase, 0.08g / L DNase I and 15% fetal bovine serum.
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Abstract
The invention discloses a method for extracting and culturing amniotic stem cells. The method comprises the steps of soaking a cleaned healthy amnion mechanically separated from a placenta into a first mixed solution for 0.5-1 hour, cleaning the healthy amnion with a trimethylolaminomethane buffer solution, scraping the surface of the amnion slightly with a cell scraper to remove epithelial cells,flushing the amnion with PBS 3-5 times, and mechanically shearing the amnion; adding amnion fragments into a second mixed solution, and digesting the amnion fragments until the amnion fragments basically disappear; superposing 1/2X ml of an amnion digestion suspension on a separated solution, performing centrifuging for 30 minutes at the speed of 1,500-2,000 rpm, and sucking a second suspension layer; finally, putting the second suspension layer into a culture medium for resuspension, and inoculating an obtained suspension into a cell culture dish for culturing at the constant temperature. According to the method, the efficiency of extracting the amniotic stem cells is higher, and the purity is higher.
Description
technical field [0001] The invention belongs to the technical field of stem cell extraction and cultivation, and in particular relates to a method for extracting and culturing amniotic membrane stem cells. Background technique [0002] With the development of tissue engineering, tissue engineered bone is expected to become a new option for bone graft or bone substitute. Seed cells are one of the three basic elements of tissue engineering, and the selection of appropriate seed cells plays a key role in bone tissue engineering. Currently, the most widely used seed cells in the field of bone tissue engineering are osteoblasts (OB), bone marrow mesenchymal stem cells (BMSCs) and periodontal ligament stem cells (PDLSCs). However, when it is obtained, there are disadvantages such as greater damage to patients, limited amount of materials obtained, and high immunogenicity of cells, which limit its clinical application and development. At the same time, the role and mechanism of s...
Claims
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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00
Inventor 王羽立杜一飞卞一峰严子欣郝舒姝刘柯玥李轩徐帆
Owner AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV
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