Separation, purification and identification methods of human amnion mesenchymal stem cells
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A kind of stem cell, separation and purification technology, applied in embryonic cells, germ cells, animal cells, etc., can solve the problems of lack, inability to distinguish hAMCs and amniotic epithelial cells, cumbersome cost, etc., to achieve the effect of simple method
Inactive Publication Date: 2012-07-11
AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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Problems solved by technology
At present, there is not only a lack of unified identification standards for hAMSCs, but also many problems: ① The tested samples are inconsistent, and the phenotypic markers of primary and passaged cells are different; SSEA-4, OCT-4, etc. Negative markers also have intersections such as CD14, CD34, CD45, HLA-DR, etc. The detection of these markers alone cannot distinguish hAMCs from amniotic epithelial cells; ③Some detection methods such as in situ immunology Histochemical staining and immunofluorescence staining cannot reflect the marker characteristics of cell populations, and some detection methods such as PCR are cumbersome and costly
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Embodiment 1
[0021] The isolation of human amniotic membrane mesenchymal stem cells of the present invention includes the following steps:
[0022] First, collect the placenta from the human term cesarean section aseptically, mechanically peel the amniotic membrane from the placental tissue, wash it several times with D-Hank’s solution to remove residual blood, and cut the amniotic membrane into 2mm×2mm fragments;
[0023] Second, add the 0.05% trypsin digestion solution containing 0.02% EDTA to the amniotic membrane fragments, rotate and digest at 37°C, 200 rpm for 10 minutes, discard the supernatant; add the digestion solution again, at 37°C, 200 rpm Rotate and digest for 30 minutes, discard the supernatant, and save the undigested amniotic membrane fragments;
[0024] Third, wash the undigested amniotic membrane fragments with D-Hank's solution, add 0.75 mg / ml collagenase containing 0.075 mg / ml DNase I, and rotate the digestion at 37°C at 200 rpm for about 2 hours until the tissue is completel...
Embodiment 2
[0027] The method for identifying human amniotic mesenchymal stem cells of the present invention includes the following steps:
[0028] First, immunocytochemical staining: hAMSCs slides were rinsed with PBS buffer, fixed with 40 g / L paraformaldehyde for 10 min, rinsed with PBS, treated with 0.3% Triton-X100 for 15-20 min, 3% H 2 O 2 Inactivate endogenous peroxidase for 10 min, rinse with PBS, block with normal goat serum for 30 min, add vimentin antibody or mouse anti-human CK19 antibody dropwise and incubate for 1 h at room temperature, rinse with PBS, and add pika general secondary antibody dropwise Incubate for 1 hour at room temperature, rinse with PBS, develop DAB, counterstain with hematoxylin, seal with neutral gum, and observe under an optical microscope. The negative control group replaced the primary antibody with PBS. The result should be that hAMSCs express vimentin, but not the epithelial cell marker CK19, which can rule out the mixing of amniotic epithelial cells in...
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Abstract
The invention discloses separation, purification and identification methods of human amnion mesenchymal stem cells. The separation method of hAMSCs (human amnion mesenchymal stem cells) comprises the following steps: fragmentating human amnion; and carrying out two-step rotating digestion with trypsin of EDTA (ethylene diamine tetra-acetic acid) and collagenase of DNaseI, filtering with a steel mesh and collecting cell filtrate namely separated original hAMSCs. The purification method of hAMSCs comprises the following steps: incubating original hAMSCs with an LG (low glucose)-DMEM (dulbecco modified eagle medium) culture medium in a CO2 incubator; removing amnion epithelial cells which do not perform complete adherence growth under an inverted microscope; replacing a new culture medium on the third day; digesting with a trypsin-EDTA solution after cell converge degree reaches 80-90%; and collecting cells so as to obtain high-purity hAMSCs. The identification method of hAMSCs comprises the following steps: identifying hAMSCs and the amnion epithelial cells by adopting immunocytochemical staining vimentin and CK19; and detecting expressions of CD29, CD44, CD166, CD34 and CD45 by adopting a flow cytometry. The separation and purification methods disclosed by the invention have the advantages of high yield, high activity and high purity of hAMSCs; and the identification method is simple, convenient and precise.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for separating, purifying and identifying human amniotic mesenchymal stem cells. Background technique [0002] Human amniotic mesenchymal stem cells (hAMSCs) originate from the extraembryonic mesoderm in early embryos and have similar phenotypes to bone marrow mesenchymal stem cells, such as the expression of SSEA-4, OCT-4, CD29, CD44 , CD73, CD90, CD166 and vimentin, but not CD34, CD45, CD80, CD86 and HLA-DR; it has multilineage differentiation potential and can differentiate into hepatocytes, bone cells, chondrocytes and adipocytes under appropriate induction conditions Cardiomyocytes, pancreatic islet cells, neuron-like cells, etc.; have the advantages of a wide range of sources, convenient material collection, no invasive injury, and no medical ethical issues involved. Therefore, hAMSCs as donor cell resources have broad application prospects in the field of regenerative m...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735G01N33/53G01N15/10
Inventor 方宁陈代雄
Owner AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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