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Feeder-layer cells, and preparation method and application thereof

A technology of feeder cells and cells, applied in the biological field, can solve the problems of not being able to maintain stem cell self-renewal well, the laboratory does not have experimental conditions, and the price of mitomycin C is expensive, so as to achieve low cost and avoid cross-contamination , the effect of improving efficiency

Inactive Publication Date: 2017-08-22
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mitomycin C is expensive, the whole production process is complicated, wasteful and time-consuming, and there is also the problem of cell cross-contamination
Although radiation treatment can process cells in large quantities, general laboratories do not have experimental conditions and have limitations.
[0004] Although many feeder-free culture systems have been established in recent years to cultivate pluripotent stem cells, the cell substrates in these systems are relatively expensive, and some pluripotent stem cells may undergo cell differentiation and apoptosis, which cannot maintain the stem cells well. Self-renewal, therefore, cannot completely replace traditional feeder culture methods

Method used

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  • Feeder-layer cells, and preparation method and application thereof
  • Feeder-layer cells, and preparation method and application thereof
  • Feeder-layer cells, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A kind of feeder layer cell, its preparation method is as follows:

[0034] (1) Cell culture: 5×10 4 A primary isolated mouse fetal fibroblast (MEF for short) was inoculated in a 35 mm culture dish, added 2 mL of culture medium, and incubated at 37 °C, 5% CO 2 After 36 hours, when the cell density reaches 80-90%, the culture is stopped; the formula of the culture medium is: high-glucose DMEM (brand: HyClone, batch number: AB10134659) and fetal bovine serum (the brand is Gibco, batch number 548979), the volume ratio of high-glucose DMEM and fetal bovine serum is 17:3, and fetal bovine serum is referred to as FBS;

[0035] (2) Treat with methanol: Take out the culture dish from the cell culture incubator, remove the culture medium, wash the cells obtained in step (1) with phosphate buffered saline (PBS for short), then remove the PBS, and pipette 1mL methanol at 4°C (methanol is pre-cooled in a refrigerator at 4°C in advance), add it to the petri dish, place it at room ...

Embodiment 2

[0038]A kind of feeder layer cell, its preparation method is as follows:

[0039] (1) Cell culture: 5×10 4 A line of mouse fibroblasts (NIH3T3 for short) was inoculated in a 35 mm culture dish, added 2 mL of culture medium, and incubated at 37 °C, 5% CO 2 After 24 hours, when the cell density reaches 80-90%, the culture is stopped; the formula of the culture medium is: composed of high-glucose DMEM and calf serum, high-glucose DMEM and calf serum The volume ratio of serum is: 9:1;

[0040] (2) Treat with methanol: Take out the culture dish from the cell culture incubator, remove the culture medium, wash the cells obtained in step (1) with phosphate buffered saline (PBS for short), then remove the PBS, and pipette 1mL methanol at 4°C (methanol is pre-cooled in a refrigerator at 4°C in advance), add it to the petri dish, place it at room temperature for 5 minutes, remove the methanol, and then let it stand on the ultra-clean workbench for 3-6 minutes to make the methanol evapo...

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Abstract

The invention provides feeder-layer cells and a preparation method thereof. The preparation method comprises the following steps: (1) cell culture: inoculating a culture dish with fibroblasts of a mouse, adding a culture solution, carrying out culture, and stopping culture when cell density reaches 80 to 90% so as to obtain cultured cells; (2) treatment with methanol: taking out the culture dish, removing the culture solution, cleaning the cultured cells with a phosphate buffer, then removing the phosphate buffer, adding methanol, carrying out standing at room temperature for 5 min, then removing methanol, carrying out standing on an ultra-clean workbench for 3 to 6 min and sealing the culture dish with a sealing membrane so as to obtain the feeder-layer cells. The invention also provides application of the feeder-layer cells to culture of multi-potent stem cells. The method has the advantages of easiness, conservation of time and low cost; and the prepared feeder-layer cells can be repeatedly used, and cross contamination can be avoided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a feeder layer cell and its preparation method and application. Background technique [0002] Pluripotent stem cells, including embryonic stem cells (ES) and induced pluripotent stem cells (iPS), have the ability of self-renewal and multilineage differentiation, so they have broad application prospects in the fields of tissue engineering and regenerative medicine. In vitro culture of pluripotent stem cells requires feeder cells as a substrate to provide nutrients. When Evans and Kaufman isolated mouse embryonic stem cells from the inner cell mass of mice in 1981, referring to the microenvironment in vivo, a layer of mouse fetal fibroblasts treated with mitomycin C was placed in a culture dish as a feeder layer. The treated mouse fetal fibroblasts lose their ability to divide and proliferate, and can provide multiple supports for embryonic stem cell attachment, proliferat...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/0735C12N5/074C12N5/077
CPCC12N5/0603C12N5/0606C12N5/0656C12N5/0696C12N2500/84
Inventor 王华岩任亚辉
Owner NORTHWEST A & F UNIV
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