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Serum-free feed layer-free mouse induced pluripotent stem cell (iPSC) induction medium and culture method using the same

A pluripotent stem cell, feeder-free technology, which is applied in the field of induction and culture of mouse induced pluripotent stem cells, can solve the problems of labor-intensive, material-intensive, cumbersome preparation of feeder cells, affecting target cells, etc., and achieves induction efficiency. Increases, maintains pluripotency and the ability to multiply rapidly, time-reducing effects

Inactive Publication Date: 2018-03-06
GUANGDONG XTEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, induce in the feeder layer system. The feeder layer cells will affect the observation of the target cell morphology. At the same time, if the feeder layer cells are not in good condition, it will directly affect the entire process of inducing iPS cells, which will affect the induction efficiency and the induction of iPS cells. time has a greater impact
At the same time, the preparation process of feeder layer cells is relatively cumbersome, which will consume a lot of manpower and material resources, and the differences between different batches of feeder layer cells cannot be resolved.
Secondly, serum is a kind of exogenous substance with quite complex components, and stem cells may die and differentiate in large quantities when cultured with medium containing fetal bovine serum

Method used

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  • Serum-free feed layer-free mouse induced pluripotent stem cell (iPSC) induction medium and culture method using the same
  • Serum-free feed layer-free mouse induced pluripotent stem cell (iPSC) induction medium and culture method using the same
  • Serum-free feed layer-free mouse induced pluripotent stem cell (iPSC) induction medium and culture method using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Serum-free, preparation of culture medium in feeder-free culture system

[0046] A serum-free, feeder-free induction culture medium for pluripotent stem cells, which can be used as an induction medium when mouse somatic cells are induced to form iPSCs, and can also be used for culturing mouse induced pluripotent stem cells. The pluripotent stem cell medium contains 15 μg / mL insulin, 25 μg / mL transferrin, 30 ng / mL epinephrine, 15% KSR, 1% non-essential amino acids, 1% L-glutamine, 1% 2-mercapto Ethanol, 1 μM CHIR99021, 3 μM PD0325901, 1000 units / ml of leukemia inhibitory factor (LIF) in DMEM medium.

Embodiment 2

[0047] Example 2 Recombinant Retrovirus Infection of Mouse Embryo Fibroblasts

[0048] 1 × 10 per well in a six-well plate 5 Cells were inoculated with mouse embryonic fibroblasts and cultured overnight in a 37°C, 5% CO2 incubator. The mOct4, mSox2, mKLF4 and mc-myc(OSKM) genes were transferred into mouse embryonic fibroblasts by pMXs retrovirus-mediated method, and the virus was infected twice, and MEF cells were infected with EGFP recombinant retrovirus as a control . After the second virus infection overnight, the cell culture medium was replaced with the above serum-free, feeder-free pluripotent stem cell culture medium to continue culturing and induction. The timing of induction of mouse somatic cell reprogramming using serum-free, feeder-free pluripotent stem cell medium is as follows: figure 1 shown. The green fluorescence of MEF cells was observed after EGFP recombinant retrovirus infected MEF cells for 48 hours, and the results were as follows: figure 2 shown. ...

Embodiment 3

[0049] Example 3 Utilizing serum-free, feeder-free system to induce mouse somatic cell reprogramming after virus infection

[0050] Observe the reprogramming process of mouse embryonic fibroblasts, see the results image 3 . in image 3 a is the cell morphology of MEF cells before infection with OSKM virus; image 3 b: On the 3rd day after MEF cells were infected by OSKM virus, some cells formed a type of cells with small cell volume and increased nuclear-cytoplasmic ratio, and aggregated and grew to form cell clusters; image 3 c: ES-like clones appeared on the 5th day after MEF cells were infected with OSKM virus; image 3 d: On the 6th day after the MEF cells were infected with OSKM virus, clones similar to embryonic stem cells appeared, with clear boundaries, and the volume of the clones gradually increased, and the diameter of the clones could reach more than 100 μm.

[0051] The results show that when mouse embryonic fibroblasts are reprogrammed in the serum-free and...

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Abstract

The invention provides a serum-free feed layer-free mouse induced pluripotent stem cell (iPSC) induction medium and a culture method using the same. The medium is a serum-free feed layer-free medium,can be used as an induction medium for mouse somatic cell-induced formation of iPSCs, improves iPS cell induction efficiency, shortens the induction time and can be used for culturing mouse iPSCs. Inthe culture system, after 20 passages of mouse iPSCs, the mouse iPSCs retain stem cell pluripotency and the normal cell karyotype. An internal teratoma experiment result shows that after culture for 20 passages, the mouse iPSCs in the culture system retain a trilaminar differentiation potential of PSCs.

Description

technical field [0001] The invention relates to the technical field of induction and cultivation of mouse induced pluripotent stem cells, in particular to a serum-free and feeder-free medium for mouse pluripotent stem cells and a method thereof. Background technique [0002] In 2006, Shinya Yamanaka's laboratory first reported the introduction of four transcription factors, Oct3 / 4, Sox2, c-Myc and Klf4, into mouse fibroblasts through retroviral vectors, and found that they could be induced to form embryos with similar A class of cells with stem cell morphology and growth characteristics, such cells are called induced pluripotent stem cells (Induced Pluripotent Stem Cells, iPSCs). Mouse induced pluripotent stem cells have unlimited proliferation potential and differentiation pluripotency. Under the conditions of in vitro culture, they can be induced to differentiate into specific types of cells (such as nerve cells, liver cells, pancreatic islet cells and muscle cells) throug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0696C12N2500/25C12N2500/32C12N2500/44C12N2501/235C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2501/81C12N2506/02
Inventor 蔡亚雄陈勇彭特刘樱于云飞乔志平
Owner GUANGDONG XTEM BIOTECH CO LTD
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