Serum-free tumor stem cell culture medium and application thereof
A tumor stem cell, serum-free technology, applied in the field of cell biology research, can solve the problem of time-consuming and high cost, and achieve the effects of high safety, good cell activity and physiological characteristics, and stable growth
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Embodiment 1
[0027] This example is the preparation method of compound 4(15)-cineole-5,7-dihydroxy-1-one
[0028] The preparation method of compound 4(15)-cineole-5,7-dihydroxy-1-one comprises the following steps:
[0029] 1) Fermentation culture of the mangrove fungus Curvularia sp.3e-GC-1CGMCC NO: KY048419: use solid rice medium for fermentation, the medium is rice: seawater = 1:1 (3% sea salt) ;Pick the cultured strains in the slant into the fermentation medium, and culture them statically at room temperature 28°C for 30 days;
[0030] 2) Extract the above-mentioned cultivated cells with chloroform / methanol 1:1 for 3 times, concentrate to obtain the extract, use ethyl acetate: water to distribute and extract the extract for 3 times, concentrate the ethyl acetate to obtain the organic phase of the cells Extract, wherein the mass ratio of ethyl acetate to water is 1:1;
[0031] 3) With petroleum ether-ethyl acetate-methanol as the elution system, the extract is roughly separated by sili...
Embodiment 2
[0037] This example is for the preparation of tumor stem cell culture medium.
[0038] The MCDB153 medium is used as the basal medium, and its composition is as follows:
[0039]
[0040]
[0041] Dissolve the above components in pyrogen-free ultrapure water and add 2 mg / L ethanolamine, 4.7 μg / L oleic acid, 4.7 μg / L linoleic acid, 5 μg / L selenium, 1.2 g / L sodium bicarbonate, and use 1N hydrochloric acid (HCl) or 1N sodium hydroxide (NaOH) to adjust the pH value to 7.4, then add 0.5mg / L albumin, 0.125μg / L epidermal growth factor, 0.33mg / L insulin-like growth factor 1, 0.33mg / L bovine pituitary extract, 10mg / L insulin, 5.5mg / L transferrin, 0.33mg / L hydrocortisone, 0.39mg / L epinephrine, 4(15)-cineole-5, 3.6mg / L of 7-dihydroxy-1-ketone, 100u / L of penicillin and 100u / L of streptomycin, the culture solution is filtered and sterilized, which is the serum-free tumor stem cell liquid medium of the present invention. The basal medium of the present invention contains nutrients r...
Embodiment 3
[0043] In this example, the tumor stem cell culture medium prepared in Example 2 was used to culture the prostate cancer Du145 cell line.
[0044] Prostate cancer stem cells can be enriched by culturing the prostate cancer Du145 cell line with the tumor stem cell culture medium of the present invention. In the experiment of this example, the prostate cancer Du145 cells were first digested into single cells, and 1×10 3 The density of cells / ml inoculated the cells into CorningTM ultra-low adherent 24-well cell culture plates, cultured them in the tumor stem cell culture medium of the present invention, and observed the cells under the microscope on the 1st, 3rd, 6th and 9th day of culture respectively The growth conditions of the obtained results are as follows figure 1 shown.
[0045] Depend on figure 1 It can be seen that the prostate cancer stem cells that form tumor microspheres (Tumor Spheroid) can be enriched by Du145 cells cultured in the stem cell medium of the presen...
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