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A method for isolating menstrual blood-derived mesenchymal stem cells
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A technology of mesenchymal stem cells and separation methods, applied in the field of menstrual blood-derived mesenchymal stem cells, can solve the problem of less MSCs, achieve good antibacterial effect, reduce mortality, and high purity
Active Publication Date: 2016-07-06
CHENGDU QINGKE BIOTECH
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At present, adult bone marrow is the main source of MSCs clinical application, but there are also some defects, such as the amount of MSCs in adult bone marrow is very small (about 0.001% 0.01%)
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Embodiment 1
[0028] Treat ten menstrual blood from different age sources, divide them evenly into two parts, mark the distribution as sample A and sample B, and separate sample A by the method provided by the present invention. The details are as follows:
[0029] A method for isolating menstrual blood-derived mesenchymal stem cells, comprising the following:
[0030] a. Collection of menstrual blood: transfer the menstrual blood to phosphate buffered saline 1-2 times the volume of menstrual blood to obtain a mixture, and store the mixture at 4°C;
[0031] The pH value of the phosphate buffer is 7.2, and the phosphate buffer includes EDTA with a mass concentration of 5 μg / ml, sodium citrate of 50 μg / ml, trehalose of 50-100 mg / ml, 0.03- 0.05mg / ml penicillin, 0.05~0.1mg / ml streptomycin, 0.05~0.1mg / ml fluconazole.
[0032] b. transfer the mixture obtained in step a within 48 hours to an equal volume of PBS buffer and mix well, then filter through a 200-mesh sieve to obtain the filtrate, and ...
Embodiment 2
[0048] Process ten parts of menstrual blood from different age sources, divide evenly into two parts, and mark the distribution as sample A and sample B, extract with traditional extraction methods and the method of the present invention (embodiment 1) respectively and from the contamination probability of samples , Extracted cell morphology and cell purity for comparison. Sample B is carried out by the traditional method, and the details are as follows:
[0049] Traditional menstrual blood-derived mesenchymal stem cell isolation methods include the following:
[0050] a. Collect menstrual blood: store menstrual blood sample A in phosphate buffered saline PBS containing 0.2ml amphotericin B and 0.2ml penicillin mixture, and store at 4°C;
[0051] b. Transfer the mixture sample A obtained in step a within 48 hours to an equal volume of PBS buffer and mix well, then slowly add it to the Ficoll lymphocyte separation medium (the volume ratio of Ficoll: sample is 1:1 ~1:2) centri...
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Abstract
The invention provides a novel menstrual blood-derived mesenchymal stem cell separation method, belonging to the field of methods for separating stem cells from menstrual blood. In order to solve the problems of long centrifugal time, relatively serious cell injury, low cell yield and the like existing in a lymph separating medium method in the traditional method, the invention provides the novel menstrual blood-derived mesenchymal stem cell separation method comprising the steps of with source-wide menstrual blood as a material, storing by using a special preserving fluid in a collecting process; then, carrying out primary separation by using a density gradient centrifugation method of a lymphocyte separation tube, and optimizing the centrifugal time and centrifugal rotating speed in separation; and next, carrying out secondary separation according to the wall attachment growth characteristic of the stem cells, simultaneously separating and amplifying the stem cells, and maintaining the activity of the stem cells to the maximum extent in the separation process. The obtained menstrual blood-derived mesenchymal stem cell is high in purity, quantity and application value. The novel menstrual blood-derived mesenchymal stem cell separation method is simple in operation and low in cost.
Description
technical field [0001] The invention relates to a method for isolating stem cells from menstrual blood, in particular to a method for isolating mesenchymal stem cells derived from menstrual blood. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) have broad application prospects in developmental biology research and clinical treatment, such as tissue and organ repair, MSCs were first confirmed in the bone marrow, and then in adult peripheral blood, periosteum, muscle and MSCs were found in adipose tissue and other places. MSCs belong to pluripotent stem cells, which can differentiate into different types of cells such as bone, fat, cartilage, muscle and liver cells under the corresponding induction conditions. At present, adult bone marrow is the main source of MSCs clinical application, but there are some defects, such as the amount of MSCs in adult bone marrow is very small (about 0.001% 0.01%). Furthermore, under aging and disease condi...
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