Method for preparing biological material with stem cell activity
A stem cell and stem cell technology, applied in the field of biological regeneration, can solve the problems of cumbersome preparation process and complicated raw materials, and achieve the effects of strong universality, simple operation and easy acquisition.
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[0032] The invention provides a method for preparing a biofilm, comprising:
[0033] Place the stem cells in the culture medium, and when the density of the stem cells reaches 70-85%, add plasma to the culture medium, and continue to culture for 20-30 hours to obtain a biofilm;
[0034] Wherein, the volume ratio of the added plasma to the culture medium is 1:2-1:6.
[0035] The preparation method of the biofilm provided by the present invention is prepared by pure biological cell technology, and plasma is added in the process of stem cell culture, and the volume ratio of the added plasma to the culture medium is 1:2-1:6, and the plasma contains fibrinogen , Fibrinogen will form water-insoluble fibrin under the action of thrombin, and fibrin will undergo cross-linking reaction to form an insoluble and stable fibrin film. At the same time, the fibrin film will form a complex with stem cells during the cross-linking reaction, thereby preparing a stem cell biomatrix film that can...
Embodiment 1
[0055]Example 1 In Vitro Isolation and Culture of Mesenchymal Stem Cells
[0056] Mesenchymal stem cells are extracted from the umbilical cord tissue of healthy people, and the obtained mesenchymal stem cells are cultured and expanded in vitro. After the primary cells were collected, they were subcultured at a ratio of 1:3. When the cells covered the bottom of the bottle with a density of about 80%, they were digested and collected with trypsin, and subcultured at the same ratio, up to the fourth generation. Collect the cells of the 4th generation. The collected cells have stable biological properties, good cell activity and strong proliferation ability. The specific process is as follows:
[0057] 1) Take the umbilical cord of a healthy person;
[0058] 2) Remove the blood vessels and outer amniotic membrane in the umbilical cord, and keep Wharton's jelly;
[0059] 3) Cut the umbilical cord tissue into 1-2cm 2 small pieces;
[0060] 4) Inoculate the cut pieces into T25 c...
Embodiment 2
[0066] Example 2 Identification of mesenchymal stem cells and detection of differentiation potential
[0067] Identification method:
[0068] a) Morphological analysis and cell counting of mesenchymal stem cells: cells cultured to the third passage were observed under an inverted microscope and photographed. Take a small amount of cell suspension and count the number of cells.
[0069] b) Phenotype determination by flow cytometry: cells of passage 3 were collected, digested with 0.25% trypsin and collected, centrifuged and washed twice with normal saline. Add mouse IgG-FITC and IgG-PE to the isotype control tube, and add mouse anti-human CD to the detection tube 105 -PE, CD 90 -FITC, CD 73 -PE, CD 45 -FITC, CD 34 -PE HLA-DR-PE 5 μl. Incubate at room temperature in the dark for 30 min, and detect by flow cytometry.
[0070] Identification results and cell activity analysis:
[0071] Under an inverted microscope, the morphology of the third-generation mesenchymal stem c...
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