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Stem cell culture medium and method for culturing endometrium stem cells

An endometrial stem cell and cell culture technology, applied in the field of stem cell culture medium, can solve the problems of high price, easy aging of cells, and many medium components, and achieve low cost, maintain activity and stemness, and fast cell proliferation efficiency. Effect

Inactive Publication Date: 2016-05-18
HANGZHOU S EVANS BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Aiming at the problems that the existing culture medium has many components, is expensive, and the cells are easy to age with the increase of the expansion generation, the present invention provides an economical and effective stem cell culture medium, which can stably expand stem cells and maintain their activity and stem cells for a long time. At the same time, it provides a culture method that effectively promotes the growth of stem cells, and obtains a stable and large number of stem cells by collecting, separating, and co-culturing menstrual blood and uterine exfoliated endometrium separately

Method used

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  • Stem cell culture medium and method for culturing endometrium stem cells
  • Stem cell culture medium and method for culturing endometrium stem cells
  • Stem cell culture medium and method for culturing endometrium stem cells

Examples

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Embodiment 1

[0034] Stem cell medium, according to the volume content of the total volume percentage, low-sugar DMEM medium accounted for 82%, fetal bovine serum accounted for 15%, penicillin and streptomycin double antibody solution accounted for 1%; gentamicin sulfate accounted for 1%; glutamine Amide accounts for 1%.

Embodiment 2

[0036] The method for cultivating endometrial stem cells using the stem cell medium provided in Example 1 comprises the following steps:

[0037] Step 1: Use the menstrual blood collection device to collect menstrual blood and endometrial tissue, and transfer the menstrual blood to a sterile centrifuge tube containing sodium heparin, transfer the endometrial tissue to a sterile culture dish, and use a sealing film to separate the centrifuge tube and culture Seal the opening of the dish, and transport it to the laboratory at low temperature (4-8°C) within 24 hours;

[0038] Step 2: Fully mix the menstrual blood in the centrifuge tube described in step 1 with an equal volume of normal saline, which contains 1% penicillin and streptomycin double antibody solution for inhibiting bacterial growth. Mix menstrual blood and normal saline evenly, superimpose with Ficoll separation solution 1:1, centrifuge at 800g for 20min, and collect buffy coat mononuclear cells. Then resuspend the ...

Embodiment 2

[0043] The endometrial stem cell identification method described in Example 2 is as follows:

[0044] 1) Morphological characteristics: Place the cell culture flask under an inverted microscope, connect the microscope to a camera device, adjust the field of view of the cells and take pictures. The cells are in the shape of long spindles and arranged in clusters.

[0045] 2) Draw the cell growth curve: resuspend the cells after subculture to obtain a cell suspension, and add the suspension evenly to a 1-well plate; start counting the cells at 24 hours, count once every 12 hours thereafter, and take 3 wells of cells each time, Count them separately, and take the average value of 3 wells for the counting results, and count continuously for 6 days. According to the cell counting results, the growth curve was drawn with unit cell number (cell number / ml) as the ordinate and time as the abscissa.

[0046] 3) Detection of cell surface markers: Directly label cell surface molecules wi...

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Abstract

The invention provides a stem cell culture medium and a method for culturing endometrium stem cells by using the stem cell culture medium. The method comprises the following steps: separately collecting menstrual blood and endometrium tissues, respectively culturing the menstrual blood and endometrium tissues in the stem cell culture medium provided by the invention to respectively obtain menstrual blood adherent cells and endometrium adherent cells, culturing the menstrual blood adherent cells and endometrium adherent cells in a cell culture bottle, collecting the adherent cells by trypsinization, inoculating the adherent cells in a cell coculture dish, and culturing the adherent cells in the stem cell culture medium provided by the invention. The stem cell culture medium has the advantages of simple components, fewer added components and lower cost. After more than 20 generations of in-vitro culture, the cells can not easily have the phenomenon of aging or degeneration, and can maintain the activity and stem property of the stem cells for a long time. The stem cell culture method is simple and effective, the cell proliferation efficiency is high, and the in-vitro culture doubling time is only 20 hours or so. The cells can be stably amplified by 50 generations.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for isolating and cultivating endometrial stem cells from menstrual blood and the used stem cell culture medium. Background technique [0002] A large number of studies have proved that endometrial stem cells have strong regenerative ability, multi-differentiation potential and low immunogenicity. They can be used as seed cells for cell therapy and tissue engineering, and their application prospects are broad. Endometrial stem cells were first extracted from curettage tissue. With the deepening of research, people found that stem cells with regenerative ability can also be isolated from women's menstrual blood, and the acquisition method is non-invasive and has no ethical issues. The method is simpler and more convenient. Safety. The endometrial stem cells obtained from menstrual blood have a strong regenerative ability, and its main performance is: rapid expans...

Claims

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Application Information

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IPC IPC(8): C12N5/074A01N1/02
CPCC12N5/0682A01N1/0221C12N2500/32C12N2500/84
Inventor 戴玲华戴唯悠
Owner HANGZHOU S EVANS BIOSCI LTD
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