Cell culture layer and application in culture of human primary cancer cells thereof

A feeder layer and cell technology, applied to animal cells, tumor/cancer cells, vertebrate cells, etc., can solve problems such as difficult operation, difficult to successfully separate and cultivate human tumor stem cells, and difficult to grasp the titer concentration of mitomycin, etc. To achieve the effect of promoting growth

Inactive Publication Date: 2014-07-02
山东大学附属千佛山医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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
[0003] The use of feeder layer in cell culture: There are foreign reports using J2 (a subtype of 3T3 cell) cells as a feeder layer for primary culture of tumor cells, but there is no mention of using it 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 easy to operate, and is currently not available in China.
[0004] Currently commonly used feeder layer preparation methods: Co60 or mitomycin is often used to treat cells to prepare feeder layers, which has the following defects: it is difficult to find Co60 radioactive sources in China at present; use mitomycin to treat feeder layers, different batches of silk from different It is difficult to grasp the titer concentration of lysomycin, and there are drug-resistant cells, and even cause malignant transformation of fibroblasts

Method used

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  • Cell culture layer and application in culture of human primary cancer cells thereof
  • Cell culture layer and application in culture of human primary cancer cells thereof
  • Cell culture layer and application in culture of human primary cancer cells thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of cell feeder layer

[0042] (1) Determination of X-ray dose:

[0043](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.

[0044] (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).

[0045] (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 irradiation ...

Embodiment 2

[0069] Example 2 Culture of primary colon cancer cells on cell feeder layer

[0070] (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:

[0071] Use DMEM to culture BALB3T3 cells based on 6-well plates, culture the cells adherently until 70% to 80% of the culture flasks are covered, and select the culture flasks with good cell growth status for irradiation (according to the concentration of 30% of the bottom area, the general bottom area 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); the irradiated cells ar...

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Abstract

The invention discloses a cell culture layer which is prepared by the following methods: culturing BALB3T3 cells by adopting a DMEM culture medium, performing cell adherent culture until 70-80 percent of a culture bottle is full, and irradiating; performing X-ray irradiation by taking 3000cGy as a radiation dose; culturing by using an improved F culture medium after irradiation; culturing for 8 hours, and obtaining the culture, namely the cell culture layer. The cell culture layer can be used for culturing human primary cancer cells or cancer stem cells. The invention also discloses improvement of cell types of the cell culture layer as well as a preparation method of the cell culture layer. Compared with J2 cells, the 3T3 cells for preparing the culture layer are easily obtained, and the cell concentration and state can be easily controlled in the culture process. When the cell culture layer is used for culturing the human primary cancer cells or cancer stem cells, growth of the primary cancer cells can be well promoted, and human cancer stem cells in a higher ratio are obtained (the number of CD44 positive cells is improved by about 80 percent).

Description

technical field [0001] The invention relates to a cell feeder layer, a preparation method thereof, and an 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: There are foreign reports that use J2 (a subtype of 3T3 cell) cells as a feeder layer for primary culture of tumor cells, but there is no mention of using it 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 lay...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/09C12N5/095
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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