A method for preparing feeder layer cells

A technology of feeder cells and adherent cells, which is applied in the field of preparation of feeder cells, can solve the problems of cumbersome preparation process, cell treatment methods and conditions without exact procedures and reports, and heavy workload, so as to simplify experimental operations, Overcoming counting subjectivity and inaccuracies and supporting growth effects

Inactive Publication Date: 2011-12-14
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the current methods for isolating and culturing feeder cells are empirical, the preparation process is cumbersome, and the workload is heavy. There are no exact regulations and reports on the methods and conditions of cell processing.

Method used

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  • A method for preparing feeder layer cells
  • A method for preparing feeder layer cells
  • A method for preparing feeder layer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, preparation and application effect of feeder layer cells

[0044] 1. Preparation of feeder cells

[0045] 1. Preparation of mouse embryonic fibroblasts (primary cells)

[0046] (1) Take a 13.5-day-old ICR pregnant mouse, kill it by breaking the neck, soak it in alcohol for a few seconds to disinfect, control the alcohol and place it in a sterile petri dish; expose the uterus under sterile conditions, lift the end of the cervix near the cervix with tweezers, The mesentery was separated, and the uterine horns were cut off; the whole uterus was taken out, placed in a plate with PBS buffer, washed three times with PBS buffer, and the residual blood on the surface was discarded.

[0047] (2) Cut the uterus along the side of the mesentery, take out the embryo with the fetal membrane, put it in another plate filled with PBS buffer, wash it thoroughly, discard the surface red blood cells; tear the fetal membrane with small forceps, take out Fetal mice, discard t...

Embodiment 2

[0071] Embodiment 2, preparation and application effect of feeder layer cells

[0072] 1. Preparation of feeder cells

[0073] Mouse embryonic fibroblasts were purchased from Sciencell, USA (article number M7540).

[0074] 2. Passage of mouse embryonic fibroblasts

[0075] During the passage process, the cells were cultured with complete medium, and the passages were carried out in 100mm culture dishes.

[0076] (1) Culture mouse embryonic fibroblasts (P0 generation cells), observe the adherence situation, and the cell density reaches about 90% after 3 days.

[0077] (2) The cells in step (1) are digested and subcultured at 1:5, which is the P1 generation, and the cell density reaches about 80% after 3 days of culture.

[0078] (3) The cells in step (2) are digested and subcultured at 1:5, which is the P2 generation, and the cell density reaches about 80% after 3 days of culture.

[0079] (4) The cells in step (3) are digested and subcultured at 1:5, which is the P3 genera...

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Abstract

The invention discloses a method for preparing feeder layer cells. The method provided by the invention comprises the following steps: (1) treating the isolated fibroblasts with 10 μg / ml mitomycin C, collecting the culture supernatant and adherent cells respectively; (2) treating the isolated fibroblasts The cells are treated with the culture supernatant collected in the previous step, and the adherent cells are collected; between the step (1) and the step (2), the following steps of 0 to 2 times are also included: the isolated fibroblast The cells are treated with the culture supernatant collected in the previous step, and the culture supernatant and adherent cells are collected respectively; the adherent cells obtained in each of the above steps are feeder layer cells. The method for preparing feeder layer cells provided by the invention has simple flow, economical and reliable effect. Human embryonic stem cell culture experiments show that the feeder layer cells prepared by the method can be passed on for a long time, and can effectively support the growth of human embryonic stem cells and keep them in an undifferentiated state.

Description

technical field [0001] The invention relates to a method for preparing feeder layer cells. Background technique [0002] The feeder layer cells are a single layer of cells prepared after specific cells (such as skin fibroblasts, fallopian tube epithelial cells, etc.) are treated with radiation or mitomycin-C to block mitosis. The feeder layer cells can secrete fibroblasts. Growth factors, insulin-like growth factors and other mitogenic factors can promote the proliferation of embryonic stem cells (Embryonic stem cells, ES cells), and can also secrete leukemia inhibitory factor (Leukemia inhibitory{actor, LIF) and other cell differentiation inhibitors to inhibit ES cells. differentiation. [0003] ES cells are a highly undifferentiated cell line with multiple developmental potentials that is isolated from the inner cell mass of embryos or the primordial genital ridge, and obtained by inhibiting differentiation in vitro. ES cells have extremely strict requirements on in vitr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/073C12N5/0735
Inventor 乔杰闫丽盈于洋李蓉严杰张妍靳红艳
Owner PEKING UNIV THIRD HOSPITAL
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