The invention provides a separation and preparation method of
dental pulp stem cells. The method comprises the following steps: using dental stem cells (DSC) separated from dental pulp tissues of human permanent teeth or
deciduous teeth; obtaining a single-
cell suspension through a double-
enzyme digestion method; washing the single-
cell suspension and the
phosphoric acid buffer
salt solution with the same volume for three times; carrying out
adherent culture on the undigested tissues; culturing in an improved MEM culture medium (MEM-alpha) supplemented with fetal calf serum (FBS) with the
volume fraction of 20%,
penicillin with the
volume fraction of 100 g / ml and
streptomycin with the
volume fraction of 100 g / ml in an
incubator at the temperature of 37 DEG C under the condition of 5% CO2; according to the double-
enzyme digestion method, the culture medium is replaced every two days; the culture medium is replaced once every 4-5 days by the tissue adherence method; after 90% confluence is achieved, performing
trypsin digestion to harvest adherent cells; under the condition, the content of CD117 positive cells exceeds 96.67%. The
dental pulp stem cells obtained by the method do not induce premature differentiation of cells, the
cell proliferation speed is greatly increased, more sufficient
nutrition is provided for the cells, synthesis of
protein is promoted, proliferation of the cells is promoted, and differentiation of the cells is inhibited; the separation speed and the separation effect are improved, and the operation difficulty is also reduced. By utilizing the method disclosed by the invention, a rapid and stable
separation method and an efficient amplification
system can be established, and the method has a very wide clinical application prospect in the fields of life science and
medicine. Along with continuous development of research, hDPSCs can play a greater role in the medical field.