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A method for preparing feeder layer cells by suspension-adherence method

A technology of feeder layer cells and adherent cells, applied in the field of in vitro cell culture, can solve the problems of low feeder quality, difficult to grasp the optimal processing time, insufficient inhibition of cell mitosis, etc., achieving flexible and convenient preparation time and improving mitotic efficiency. , the effect of fast sticking to the wall

Active Publication Date: 2017-11-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for preparing feeder cells by a suspension-adherence method for the deficiencies in the prior art; this method can overcome the insufficient inhibition of cell mitosis and the resulting The quality of the feeder is not high, the MEFs proliferation speed is too fast, the optimal processing time is not easy to grasp and other technical defects; the method of the present invention can not only fully inhibit cell mitosis, but also screen out cells in good condition as the feeder, which can improve the quality of the prepared feeder, The preparation time is more flexible and convenient

Method used

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  • A method for preparing feeder layer cells by suspension-adherence method
  • A method for preparing feeder layer cells by suspension-adherence method
  • A method for preparing feeder layer cells by suspension-adherence method

Examples

Experimental program
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Effect test

Embodiment 1

[0048] A method for preparing feeder cells by a suspension-adherence method, comprising the following steps:

[0049] (1) CF-1 MEFs primary (P0) acquisition:

[0050] Take a CF-1 mouse at 12.5 days pregnant, kill it by neck dislocation, take out the embryo under aseptic conditions, wash it with 10ml PBS and put it in a sterile petri dish; remove the head and viscera of the embryo, and wash it twice with 10ml PBS; Transfer the remaining tissue to a new sterile petri dish and cut it into pieces with sterile scissors, add 1ml of digestion solution to each embryo, pipette several times at the same time, put it in 37°C, 5% CO 2 Digest in a cell incubator for 5 minutes; use a culture solution equal to the volume of the digestion solution to terminate the digestion, blow and beat sufficiently to separate as single cells as possible; after mixing the above cell suspension, add the cell suspension of 2 embryos to each T75 culture flask and 13ml of culture solution, mix thoroughly and ...

Embodiment 2

[0069] Comparison test of feeder cells prepared by suspension-adherence method and traditional method:

[0070] Traditional methods include the following steps:

[0071] (1) CF-1 MEFs primary (P0) acquisition:

[0072] Take a CF-1 mouse at 12.5 days pregnant, kill it by neck dislocation, take out the embryo under aseptic conditions, wash it with 10ml PBS and put it in a sterile petri dish; remove the head and viscera of the embryo, and wash it twice with 10ml PBS; Transfer the remaining tissue to a new sterile petri dish and cut it into pieces with sterile scissors, add 1ml of digestion solution to each embryo, pipette several times at the same time, put it in 37°C, 5% CO 2Digest in a cell incubator for 5 minutes; use a culture solution equal to the volume of the digestion solution to terminate the digestion, blow and beat sufficiently to separate as single cells as possible; after mixing the above cell suspension, add the cell suspension of 2 embryos to each T75 culture flas...

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Abstract

The invention discloses a method for preparing feeder layer cells by a suspension-adhesion method. The method comprises the following three steps: obtaining a CF-1 MEFs primary generation; carrying out subculture of the CF-1 MEFs and preparing a CF-1 MEF Feeder. The CF-1 MEF Feeder is prepared by using the suspension-adhesion method which comprises the following steps: digesting the third generation of the CF-1 MEFs cells to form a single-cell suspension; then, bespreading to a culture bottle / vessel at the density of bespreading the culture bottle / vessel after the cells are adhered to the wall; adding MMC for treating for 0.5-3.5 hours after adhesion treatment in a culture box; and quickly absorbing a MMC-containing culture liquid and non-adhered cells in the culture vessel and just recovering adhered cells as the Feeder. The method disclosed by the invention not only can be used for fully inhibiting mitosis, but also can be used for screening cells in good states as the Feeder, so that the quality of the Feeder is improved. Meanwhile, the method can be used for overcoming the defect that the optimum treatment time is hardly mastered as the proliferation speed of MEFs is too fast, so that the preparation time of the CF-1 MEF Feeder is flexible and convenient.

Description

technical field [0001] The invention relates to a method for preparing feeder cells, in particular to a method for preparing feeder cells by a suspension-adherence method, and belongs to the technical field of in vitro cell culture. Background technique [0002] Induced pluripotent stem cells (iPSCs) have many characteristics similar to embryonic stem cells and can differentiate into almost all adult cells. Since they can be obtained from terminally differentiated adult cells through cell reprogramming technology, they can be obtained Patient-specific or disease-specific iPSCs have made them a hotspot in the study of disease pathogenesis, individualized drug therapy, drug screening and cell therapy, further shortening the distance between stem cells and clinical disease treatment. iPSCs are prone to differentiation and karyotype instability during long-term culture, and it is extremely challenging to maintain the normal undifferentiated state and self-renewal ability of iPSC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
Inventor 李玉林战立香池光范张丽红李莉莎辛颖何旭石英爱李美英吕爽贺霞石旭孙美玉刘小梅
Owner JILIN UNIV
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