Method for induced differentiation of liver cells by using endometrium stem cells
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A technology of endometrial stem cells and liver cells, applied in the field of using endometrial stem cells to induce differentiation of liver cells, to achieve the effect of enriching the source of cells
Active Publication Date: 2013-06-26
HANGZHOU S EVANS BIOSCI LTD
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Embodiment 1
[0064] Example 1. Isolation, cultivation and expansion of menstrual blood mesenchymal stem cells:
[0065] Ten menstruating women of different ages were recruited to donate menstrual blood samples on a completely voluntary basis. After 24 hours of antibiotic treatment, microbiological testing and infectious disease pathogen safety testing were carried out. After centrifugation, the supernatant was removed, and then inoculated, using Chang's general medium in 5% CO 2 , cultured in an incubator at 36-38°C (for example, 37°C) with 95% saturated humidity. Change the medium after 5-7 days to remove non-adherent cells, and then change the medium every 2-4 days (for example, 3 days). Once the cells grew to 80% confluence, they were passaged by trypsin / EDTA digestion. Then use flow cytometry to identify the molecular phenotypes of CD117, CD34, CD44, CD45, CD73, CD90, CD105, CD29, HLA-DR, etc. on menstrual blood mesenchymal stem cells.
[0066] The specific operating procedures are...
Embodiment 2
[0130] Embodiment 2, in vitro induced differentiation and detection:
[0131] The specific operating procedures are as follows:
[0132] 1. Directed induction of endometrial stem cells to differentiate into liver cells in vitro
[0133] 1) Take the third-generation cells in good growth condition, which are characterized by vigorous growth, large cell bodies, clear nuclei, rich cytoplasm, and strong refraction under the microscope, and digest them with trypsin-EDTA digestion solution (0.25%) Under the microscope, when the cells become a single round shape, stop the digestion with DMEM medium containing 10% (volume concentration) fetal bovine serum, gently pipette and centrifuge to obtain cell pellets, PBS (PBS washing solution without calcium and magnesium ions) Wash 2 times (the purpose is to wash away trypsin, fetal bovine serum and other substances).
[0134] 2) Inoculate the above-mentioned cell pellet after washing with PBS in a 6-well plate for induction culture, and inoc...
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Abstract
The invention discloses a method for induced differentiation of liver cells by using endometrium stem cells. The method comprises the following steps of: (1) carrying out isolated culture and amplification on the endometrium stem cells; and (2) carrying out in vitro induced differentiation: A, carrying out trypsinization on cells with good growth conditions of 2-4 generations, which are obtained in the step (1), ending the digestion by using a DMEM (dulbecco's modified eagle medium) culture medium containing fatal bovine serum of which the volume concentration is 9-11% when the cells, seen from a microscope, become single round cells, centrifuging, and cleaning the obtained cell deposit by using a PBS (phosphate buffered solution); B, inoculating the cell deposit which is obtained in the step A and cleaned by using the PBS to a six-hole plate to carry out induced culture; C, carrying out induced culture by selecting a system 1 adopting an induced culture medium I or a system 2 adopting an induced culture medium II; and D, weighing and changing the solution once every 2 to 3 and a half days, and observing the condition of the cells under an inverted microscope to obtain liver cell like cells with functions of the liver cells, wherein the induction time is 20-22 days.
Description
technical field [0001] The invention relates to a method for inducing differentiation of liver cells using endometrial stem cells. Background technique [0002] Orthotopic liver transplantation is the gold standard for the treatment of end-stage liver disease. However, the shortage of liver sources has become the biggest bottleneck in the clinical application of hepatocyte transplantation. Therefore, finding a suitable and abundant source of hepatocytes is a hot spot in the field of biology. In 1981, Evans et al. established the first mouse ES cell line and used it as a model. Studies have confirmed that mouse ES cells can not only be induced to differentiate into hepatic progenitor cells in vitro, but also can be further differentiated into functional hepatocytes. Animal experiments have shown that when hepatocytes differentiated in vitro are transplanted into a liver injury model, the transplanted cells can fuse to the liver tissue and alleviate the liver injury in mice,...
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