Mice-induced pluripotent stem cell induction culture medium without animal exogenous components

A technology of pluripotent stem cells and induction medium, which is applied in the field of stem cells and biology, can solve the problems of iPS purity induction efficiency, hinder iPS cell research and transplantation, etc.

Inactive Publication Date: 2018-04-20
GUANGDONG XTEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on iPS still faces two major problems, namely, the purity of the obtained iPS and the induction efficiency.
Conventional iPS induction is carried out on the feeder layer, and animal exogenous components such as fetal bovine serum are added to the induction medium. The efficiency of somatic cell reprogramming is generally lower than 1%. These problems seriously hinder the further development of iPS cells. Applications such as research and transplantation

Method used

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  • Mice-induced pluripotent stem cell induction culture medium without animal exogenous components
  • Mice-induced pluripotent stem cell induction culture medium without animal exogenous components
  • Mice-induced pluripotent stem cell induction culture medium without animal exogenous components

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, preparation of induction medium for induced pluripotent stem cells

[0030] The mouse iPSCs induction medium of the present invention uses DMEM as the base medium, and each 500mL induction medium also includes the following components:

[0031]

[0032]

[0033] The components of the induction medium were fully mixed in a grade 2 biological safety cabinet, then subpackaged, and stored at -20°C, with a validity period of 10 months. Store at 4°C when used, and it will be effective within two weeks.

Embodiment 2

[0034] Embodiment 2, the acquisition of MEF cells

[0035] MEF cells were purchased from Shanghai Cell Bank, and after quality inspection, the cell morphology was as follows: figure 1 shown. It can be seen from the figure that the normally cultured MEF cells have clear boundaries between cells and are spindle-shaped, which is a typical morphology of fibroblasts.

Embodiment 3

[0036] Example 3. Induction of mouse iPSCs

[0037] The present invention prepared the mouse iPSCs induction medium of Example 1 and an equal volume of conventional pluripotent stem cell induction medium as controls.

[0038] Induction of mouse iPSCs involves the following steps:

[0039] First, four pluripotency genes (Oct4, Sox2, Klf4, and c-Myc) were introduced into MEF cells by the following method:

[0040] 1) Inoculate MEF cells on a 6-well plate at a density of 1*10^5 / well, one of the wells has no feeder layer, and the other well is plated with feeder layer cells at a density of 2.5*10^5 one day in advance;

[0041] 2) After 1 day, mix 0.5 mL of each virus containing four pluripotency genes, and add 0.5 mL of antibiotic-free MEF medium to mix;

[0042] 3) Add polybrene so that the final concentration is 5 μg / mL;

[0043] 4) Discard the culture medium in the 6-well plate, wash 2-3 times with PBS, and add the mixture of virus and culture medium to the 6-well plate for ...

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Abstract

The invention relates to a culture medium which can quickly and effectively induce reprogramming of mice somatic cells into pluripotent stem cells and has no animal exogenous property. The induction culture medium is composed of a DMEM base culture medium, NEAA, KSR, LIF and a variety of small molecules and inorganic salts; the culture medium has clear components and has no animal exogenous pollution. The induction culture medium has the advantages of high induction efficiency, short induction time and the like, and is of great significance for development of obtaining of a large number of induced pluripotent stem cells and promotion of clinical application of the stem cells.

Description

technical field [0001] The invention relates to the field of stem cells and biotechnology, in particular, the invention relates to a mouse induced pluripotent stem cell induction medium without animal exogenous components. Background technique [0002] In 2006, Yamanaka of Kyoto University in Japan used viral vectors to transfer four transcription factors Oct4, Sox2, Klf4 and c-Myc into mouse embryonic fibroblasts and tail skin fibroblasts, and obtained clones with morphology and growth characteristics similar to ES cells. Named as induced pluripotent stem cells, and thus won the 2012 Nobel Prize in Physiology or Medicine. Since then, scientists from different countries have used similar methods and used retroviruses as vectors to introduce four transcription factors into different cells of different species, and all obtained induced pluripotent stem cells. In subsequent studies, mice with reproductive ability were obtained by injecting iPS cells into blastocysts, which fur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0696C12N2500/12C12N2500/32C12N2500/34C12N2500/44C12N2501/115C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2506/02C12N2510/00
Inventor 刘樱陈勇蔡亚雄彭特于云飞乔志平
Owner GUANGDONG XTEM BIOTECH CO LTD
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