Human umbilical cord blood mesenchymal stem cell extract and preparation method and application thereof
A technology of stem cells and human umbilical cord blood, which is applied in the field of biomedical stem cells and tissue engineering, can solve the problem of single active ingredient and achieve the effect of accelerating the reproduction rate and increasing the yield
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[0035] Example 1 Isolation and culture of human umbilical cord blood mesenchymal stem cells
[0036] 1.1 Cord blood collection
[0037] With the assistance of the obstetrics and gynecology hospital that has a long-term cooperation with our company, cord blood will be collected from cesarean women who are healthy under 30 years old, have no genetic diseases, no infectious diseases, and have negative anti-HIV and hepatitis B serum markers. . After collection, the cord blood was placed in an ice box and immediately transported to the laboratory for separation and processing. Among them, the parturients involved knew and agreed to the experimental protocol of the separated cord blood.
[0038] 1.2 Isolation and culture of mesenchymal stem cells from cord blood
[0039] The collected umbilical cord blood is diluted in an equal volume with phosphate buffered saline (PBS for short), and then the lymphatic separation solution is used for density gradient centrifugation to collect the albugi...
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[0041] Example 2 Identification of cell types of isolated human umbilical cord blood mesenchymal stem cells
[0042] 2.1 Take the umbilical cord blood mesenchymal stem cells cultured in vitro to the third generation, digest, centrifuge, and adjust the cell density to 1x106 cells / ml with PBS, and divide them into multiple tubes.
[0043] 2.2 Absorb the following antibodies: ITC-anti-human-IgG FC (purchased from Biolegend, item number 409309), FITC-anti-hunman-CD44 (purchased from Biolegend, item number 338803), FITC-anti-hunman-CD105 (purchased from Biolegend , Article number 323203), PE-anti-human-IgGFC (purchased from Biolegend, article number 409311), PE-anti-hunman-CD14 (purchased from Biolegend, article number 367103), PE-anti-hunman-CD34 (purchased from Biolegend, article number 343505), PE-anti-hunman-CD31 (purchased from Biolegend, catalog number 303105) 5ul;
[0044] 2.3 Add the antibody to the cell suspension added to the different tubes in step 2.1, incubate for 15 minutes...
Example Embodiment
[0047] Example 3 Extraction of human umbilical cord blood mesenchymal stem cell content
[0048] 3.1 Extract stem cell content from human umbilical cord blood mesenchymal stem cell culture medium;
[0049] 3.1.1 Thaw the culture medium stored in the -20°C refrigerator collected during the cell culture process in the 4°C refrigerator;
[0050] 3.1.2 Use a low-temperature centrifuge to control the temperature. When the temperature of the low-temperature centrifuge reaches 4 degrees, transfer the thawed cell culture medium into a 50ml centrifuge tube, centrifuge at 1000r / min for 5min, and filter the supernatant with a 0.45uM filter.
[0051] 3.1.3 Transfer the filtered supernatant to a new 50ml high-speed centrifuge tube and centrifuge at 10000r / min for 20min. The supernatant liquid was discarded, the sediment at the bottom of the centrifuge tube was resuspended with 2% by volume sterile water, and filtered with a 0.45uM filter to obtain the secretory content of human umbilical cord blo...
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