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Non-feeder cell cultivation system culture medium for human embryonic stem cell and cultivation method therefor

An embryonic stem cell and feeder-free technology is applied in the field of stem cell culture to achieve the effect of low culture cost

Inactive Publication Date: 2009-07-08
CHANGSHA HUI LIN LIFE TECH
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Problems solved by technology

The feeder-free culture system disclosed in this document is: DMEM / F12, 1×N2, 1×B27, 0.1mM β-ME, 1×glutamine, fetal bovine serum was added to the medium, and the concentration of FGF2 was 20ng / ml. At the same time, Matrigel pre-plated dishes are used, which does not involve the issue of clone density

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  • Non-feeder cell cultivation system culture medium for human embryonic stem cell and cultivation method therefor
  • Non-feeder cell cultivation system culture medium for human embryonic stem cell and cultivation method therefor
  • Non-feeder cell cultivation system culture medium for human embryonic stem cell and cultivation method therefor

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Embodiment Construction

[0013] Methods and steps:

[0014] 1. Human embryonic stem cell feeder-free and exogenous cytokine-free culture medium formula (NB medium);

[0015] Utilize DMEM / F12 (11330057, DMEM NUTRIENT MIX F12, Gibco) N2 SUPPLEMENT (17502048, 100 ×, Gibco), B27 SUPPLEMENT (17504044, 50 ×, Gibco), β-ME (β-mercaptoethanol, Gibco) configuration The present invention provides Medium: DMEM / F12, 1×N2, 1×B27, 0.1 mM β-ME.

[0016] The components of each 50ML are as follows: DMEM / F12 48.5ml

[0017] N2 0.5ml

[0018] B27 1ml

[0019] β-ME 91μl

[0020] 2. Petri dish pretreatment

[0021] 1) Thaw the imported fetal bovine serum at 4°C, then aliquot into 2ml / EP tubes, and store at -20°C for later use;

[0022] 2) Thaw the serum in the EP tube at room temperature;

[0023] 3) Coat the imported FBS on a 1-well plate (0.5ml) or a 6-well plate (1.5ml), at 4°C overnight or at room temperature for at least 4 hours;

[0024] 4) Aspirate the FBS befo...

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Abstract

The present invention provides a culture system and culture medium of human embryonic stem cell without feeder cells and exogenous cytokine, composed of the following constitutions: DMEM / F12, 1*N2, 1*B27, 0.1mM beta-ME. The present invention also provides a culture method of human embryonic stem cell without feeder cells based on the culture medium. In the stem cell system under the inventive culture conditions, so long as the cloning planting density is maintained at a high level, the human embryo stem cell can maintain undifferentiated state amplification, the background differentiation phenomenon does not exist, the human embryonic stem cells can be amplified in large scale, the invention has features of low culture cost and provides a technique platform for the undifferentiated state of the human embryo stem cells.

Description

technical field [0001] The invention relates to the composition of a stem cell-free feeder cell-free culture system without exogenous cytokines. [0002] The invention also relates to a method for culturing stem cells. Background technique [0003] At present, the main culture system adopted by hESCs is co-cultivation with fibroblasts [1] , the cytokines secreted by fibroblasts play a vital role in the maintenance of embryonic pluripotency. Scholars at home and abroad have identified some cytokines that play an important role in maintaining the undifferentiated embryonic stem cells from the conditioned medium of fibroblasts. Cytokines, including the Wnt signaling pathway ligand Wnt3a [2] , but Wnt3a alone did not maintain hES in an undifferentiated state [3] , fibroblasts may also secrete other cytokines, such as FGF2, Nodal, Activin A, etc. With the establishment of feeder-free culture of human embryonic stem cells, the researchers found that the Wnt signaling pathway i...

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Application Information

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IPC IPC(8): C12N15/08
Inventor 卢光琇林戈罗树伟
Owner CHANGSHA HUI LIN LIFE TECH
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