Cell culture medium and application thereof in culturing primary human tumor cells

A medium and high-glucose medium technology, applied in the field of cell culture medium, can solve the problems of difficult operation, difficult to successfully separate and cultivate human tumor stem cells, no mention of tumor stem cell culture, etc., and achieve the effect of easy acquisition and growth promotion

Inactive Publication Date: 2014-07-02
山东大学附属千佛山医院
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Problems solved by technology

[0002] At present, the commonly used methods of isolation and culture of human tumor stem cells mostly adopt the serum-free suspension culture method, which isolates and cultures stem cells with very small content from cell lines or directly purchases stem cell lines for suspension culture, while the method of adherent culture of primary tumor cells Difficult to successfully isolate and culture human tumor stem cells
[...

Method used

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  • Cell culture medium and application thereof in culturing primary human tumor cells
  • Cell culture medium and application thereof in culturing primary human tumor cells
  • Cell culture medium and application thereof in culturing primary human tumor cells

Examples

Experimental program
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Effect test

Embodiment 1

[0040] The preparation of embodiment one cell feeder layer

[0041] (1) Determination of X-ray dose:

[0042] (1) The 6-well plate was coated with 1% gelatin, placed in a constant temperature incubator with 5% carbon dioxide at 37°C overnight, the gelatin was sucked off, and washed with PBS for more than 3 times.

[0043] (2) Inoculate BALB3T3 cells of the same concentration (purchased from Shanghai Cell Bank) into 6-well plates and culture them in DMEM cell culture medium until they cover 70% of the bottom of 6-well plates ( figure 1 ), add culture medium to each well to 8ml, and place it under 6MV low-energy X-ray for gradient irradiation (source-to-skin distance 100cm).

[0044] (3) Closely observe the growth of irradiated BALB3T3 cells to determine the optimal radiation dose. Such as Figure 2 ~ Figure 4 Shown: Among them, BALB3T3 cells with irradiation doses of 2200cGy, 2400cGy, 2600cGy, and 2800cGy gradually overgrown after 7 days ( figure 2 ); BALB3T3 cells with irra...

Embodiment 2

[0068] Example 2 Culture of primary colon cancer cells on cell feeder layer

[0069] (1) BALB3T3 cells were cultured in 6-well plates with DMEM medium (shown in Table 7), and irradiated with 3000cGy as the radiation dose. The irradiated BALB3T3 cells were replaced with modified F medium and used as a cell feeder layer after 8 hours of culture. details as follows:

[0070] Use DMEM to culture BALB3T3 cells based on 6-well plates. When the cells adhere to the wall and culture to 70% to 80% of the culture flasks, select the culture flasks with good cell growth state for irradiation (inoculate according to the concentration of 30% of the bottom area, generally the bottom area The area is 25cm 2 The required amount of cells can be reached in 2 days in the culture bottle), and X-ray irradiation is carried out with 3000cGy as the radiation dose (the instrument starts from 0 when the radiation is irradiated, and the dose reaches the required radiation dose and stops immediately); th...

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Abstract

The invention discloses a cell culture medium. The raw material formula of the cell culture medium comprises 33.33ml of DMEM/High Glucose (DMEM high-glucose culture medium), 66.67ml of HAM'S/F-12 culture medium, 3-5mg/mL of plant-origin recombinant human serum albumin, 100U/ml of penicillin, 100 micrograms/milliliter of streptomycin, 2.5 micrograms/milliliter of amphotericin B, 0.2-0.5 micrograms/milliliter of hydrocortisone, 3-6 micrograms/milliliter of insulin, 6-10ng/ml of cholera toxin B, 9-12ng/ml of human epidermal growth factor EGF, 22-25 micrograms/milliliter of adenine, and 7-9 micromoles/liter of Y-27632. The cell culture medium can be used for culturing primary human tumor cells or tumor stem cells. The cell culture medium is improved; when the cell culture medium is applied to culturing the primary human tumor cells or the tumor stem cells, the cell culture medium is capable of better accelerating the growth of the primary tumor cells and obtaining the human tumor stem cells of a higher ratio.

Description

technical field [0001] The invention relates to a cell culture medium and its application in culturing human primary tumor cells or tumor stem cells. Background technique [0002] At present, the commonly used methods of isolation and culture of human tumor stem cells mostly adopt the serum-free suspension culture method, which isolates and cultures stem cells with very small content from cell lines or directly purchases stem cell lines for suspension culture, while the method of adherent culture of primary tumor cells It is difficult to successfully isolate and culture human tumor stem cells. [0003] The use of feeder layer in cell culture: ① It has been reported abroad that J2 (a subtype of 3T3 cell) cells were used as a feeder layer for primary culture of tumor cells, but there was no mention of its use for tumor stem cell culture. Moreover, the concentration and state of J2 cells need to be strictly controlled in the process of serving as a feeder layer, which is not e...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/095
Inventor 孙青贾茹张建东岳龙涛
Owner 山东大学附属千佛山医院
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