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Culture method free of multipotential stem cell without serum and feeder layer

A technology of pluripotent stem cells and pluripotent stem cells, which is applied in the field of serum-free and feeder-free pluripotent stem cell culture, can solve the problem of contamination of pluripotent stem cell applications, the inability to guarantee cell biological safety and quality control, and increase the complexity of experiments And other issues

Active Publication Date: 2014-02-05
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, serum is an exogenous substance with extremely complex components. It is impossible to determine which components in serum affect the behavior of cells in experiments and applications. Secondly, animal-derived serum and pollutants in feeder cells will contaminate a lot. Application of competent stem cells themselves
In addition, the currently widely used adherent culture method needs to lay a substrate on the bottom of the petri dish. On the one hand, it increases the complexity of the experiment and increases the cost of cell culture; on the other hand, some animal-derived substrates such as animal gelatin (gelatin) ), laminin and matrigel cannot guarantee the biological safety and quality control of cells in clinical practice

Method used

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  • Culture method free of multipotential stem cell without serum and feeder layer
  • Culture method free of multipotential stem cell without serum and feeder layer
  • Culture method free of multipotential stem cell without serum and feeder layer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: Preparation of culture medium without feeder layer and serum-free culture system

[0073] Culture medium preparation

[0074] This application uses DMEM / F12 medium, Neurolbasal medium, N2 supplement, B27 supplement, 2-mercaptoethanol (2-Mercaptoethanol), leukemia inhibitory factor (LIF), mitogen-activated protein kinase inhibitor (Stemolecule TM PD0325901), glycogen synthase kinase inhibitor 3 (Stemolecule TM CHIR99021) to prepare.

[0075] Wherein, each component of culture medium was purchased from:

[0076] DMEM / F12 medium was purchased from Gibco, the product number is 11320-082;

[0077] Neurolbasal medium was purchased from Gibco Company, the product number is 21103-049;

[0078] N2 additive was purchased from Gibco Company, the article number is 17502-048;

[0079] B27 additive was purchased from Gibco company, and the article number is 12587-010;

[0080] 2-Mercaptoethanol was purchased from Gibco Company, the article number is 21985-23; ...

Embodiment 2

[0094] Suspension culture of embodiment 2 mouse embryonic stem cells

[0095] This example uses the OCT4-GFP mouse embryonic stem cell line (OGR1) (purchased from the University of Illinois, USA), and the pluripotency of the embryonic stem cells can be visually judged by detecting the expression intensity of the green fluorescent signal.

[0096] First, mouse pluripotent stem cell clones cultured under traditional adherent culture conditions were digested with 0.05% trypsin at 37°C for 2 minutes, and the clones were blown into single cells.

[0097] Then, the prepared single cell suspension was 1.0×10 7 L -1 ~1.0×10 8 L -1 The density was inoculated in ultra-low adsorption culture dishes, placed at 37 ° C, 5% (volume ratio) CO 2 cultured in a humidified incubator. Cells should change the medium every 48 hours. When changing the medium, collect the cell suspension into a 15ml centrifuge tube, centrifuge at 800RPM (rev / min) for 3 minutes, discard the supernatant, add fres...

Embodiment 3

[0098] Subculture of embodiment 3 mouse embryonic stem cells

[0099] The mouse embryonic stem cells OGR1 cultured for 3-4 days in Example 2 were digested and reseeded at a certain cell density in a new super-adherent dish, and N2B27 was added for complete culture and suspension culture.

[0100] specifically:

[0101] (1) Transfer the amplified spherical cell suspension in Example 2 into a 15ml centrifuge tube, collect the cell spheres by centrifugation at 800RPM, discard the supernatant, add PBS and wash again by centrifugation at 800RPM, and wash the cell spheres with 0.05% Digest with trypsin at 37°C for 2-3min.

[0102] (2) Use trypsin inhibitor (T6414, Sigma) to terminate the digestion, and pipette the cell pellet to obtain a single-cell suspension. After adding an appropriate amount of PBS, centrifuge the above cell suspension at 1000RPM, discard the supernatant, resuspend the cells with fresh medium, and press 1.0×10 7 L -1 ~1.0×10 8 L -1 The single-cell suspen...

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Abstract

The invention relates to a novel method for culturing a multipotential stem cell without serum and a feeder layer. The method is stable, reliable, and can culture the multipotential stem cell for a long time and also can maintain the self-renewing of the multipotential stem cell. The method is characterized in that a culture system with clear chemical components and without serum and the feeder layer is adopted to perform suspension culture on the multipotential stem cell. According to the morphology of cultured stem cells after 15 generations, mRNA (Messenger Ribonucleic Acid) and protein expression of multipotential marker molecule, and analysis on cell chromatin karyotype, such novel generation system can maintain the properties of the multipotential stem cell during long-term culture. In addition, the differentiative potential of the multipotentail stem cell is further verified by 15-generation suspension culture in an in-vitro embryoid body formation experiment and an in-vivo teratoma formation experiment.

Description

technical field [0001] The invention relates to a system and method for culturing pluripotent stem cells in suspension without serum and feeder layer. Specifically, the present invention relates to a system and method for suspension culture of pluripotent stem cells using a serum-free, feeder-free, and chemically defined culture system. Background technique [0002] In recent decades, the study of stem cell biology has advanced people's understanding of many basic biological issues, and has also prompted people to explore cell therapy methods for many diseases. Since Evans and Matthew Kaufman first discovered mouse embryonic stem cells in 1981, embryonic stem cell research has been the focus of attention. Since then, the acquisition of induced pluripotent stem cells (iPSCs) has brought pluripotent stem cell research to a climax. Mouse pluripotent stem cells (including mouse embryonic stem cells and iPSCs) are often used as the best cell model for studying the growth, diffe...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
Inventor 段恩奎雷晓华邓智利
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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