Preparation method of subtotipotent stem cells
A subpluripotent stem cell and cell technology, applied in the field of stem cell engineering, can solve problems such as complex methods, achieve the effect of good differentiation ability and avoid pollution
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Embodiment 1
[0034] Tissue acquisition: Place the umbilical cord of the newborn under sterile conditions and separate it within 12 hours. The placenta is taken from a full-term healthy pregnant woman. The pregnant woman’s HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-Treponema pallidum antibody, ALT, and mycoplasma All other test items were negative. After the placenta was removed from the operating table, it was immersed in 0.9% physiological saline containing antibiotics and kept at 4°C for storage;
[0035] Disinfection treatment: After ligation at both ends of the umbilical cord, soak in 75% alcohol for 1-2 minutes, wash with 0.9% normal saline for 2-3 times, and remove residual blood on the surface of the tissue; Wipe clockwise with 75% alcohol cotton and repeat; the environment may not be able to achieve complete aseptic conditions and still ensure the safety of sub-totipotent stem cell extraction. The amniotic surface, chorion, and decidua surface of the umbilical cord and ...
Embodiment 2
[0042] Example 2 prepares 0.1% collagenase, other steps are the same as Example 1.
Embodiment 3
[0043] Example 3 prepares 2% collagenase, other steps are the same as Example 1.
[0044] Result analysis
[0045] Subtotipotent stem cell surface markers
[0046] Culture the collected stem cells for 2 to 3 generations, digest and centrifuge, wash the cell pellet twice with DPBS, and take about 0.5‐1X10 6 100ul of cell suspension was placed in a flow loading tube, 15ul of primary antibody was added, after incubation at 4°C for 30min, 400ul of DPBS was added, centrifuged at 1500rpm for 6min, the supernatant was discarded, 10ul of secondary antibody was added, and after 30min of incubation at 4°C, added Centrifuge 400ul DPBS at 1500rpm for 6min, discard the supernatant, add 500ul DPBS, mix well, and test on the machine. Positive indicators: Flk1, CD73, CD90, CD105, negative indicators: CD34, HLA-DR, positive rate ≥ 95%, negative rate ≤ 2%. It shows that what we have isolated are umbilical cord and placental subtotipotent stem cells rather than other cells, see Table 2 and Fi...
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