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Human embryonic stem cells culture medium without dependent feeding cell

A culture medium and stem cell technology, applied in the field of human embryonic stem cell culture medium, can solve unseen problems

Inactive Publication Date: 2007-02-07
SHIYAN TAIHE HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Cell Culture

[0075] 1. Purpose: To directly use LD-1 in hES cell culture, observe its growth and morphological changes, and set up a positive control group and a negative control group at the same time for simultaneous comparison.

[0076] 2. Grouping and methods: Inoculate 26-generation hES cells (H1) cultured on Matrigel with conditioned medium into 4-well plates coated with Matrigel, and divide them into three groups:

[0077] The first group is cultured with conditioned medium (positive control);

[0078] The second group was cultured with LD-1 medium (i.e.: normal medium + 40ng / ml Human FGF basic + 500ng / ml Recombinant Mouse Noggin / Fc Chimera);

[0079] The third group was cultured with normal medium (negative control).

[0080] According to 0.5ml culture medium / well, the culture medium was changed once a day, passaged once every 5-7 days, a total of 6 passages.

[0081] 3. Results: The cells in the first group and the second group grew well at the s...

Embodiment 2

[0083] Example 2 Immunohistochemical detection

[0084] 1. Objective: To detect the characteristics of some human embryonic stem cells cultured with LD-1 for 6 passages by immunohistochemical method.

[0085] 2. Method

[0086] 1. Cells: Plant LD-1 cultured hES cells that have been passaged multiple times into 4 wells of a 4-well plate, and plant hES cells cultured in conditioned medium and normal medium into the other two 4-well plates respectively. In 4 holes;

[0087] 2. Fix the cells: culture the above cells until the 4th day, suck off the culture medium, add 4% paraformaldehyde, and leave it at room temperature for 10 minutes to fix the cells;

[0088] 3. Cell grouping: the aforementioned fixed cells were divided into 4 groups, and each group contained 1 well of hES cells cultured in LD-1, conditioned medium and normal medium respectively.

[0089] 4. Primary antibody: a) Oct-4 (Chemicon MAB4305, mouse IgG1), b) SSEA-4 (Chemicon MAB4304, mouse IgG3), c) TRA-1-60 (Chemi...

Embodiment 3

[0095] Embodiment 3 quantitative analysis

[0096] 1. Objective: To quantitatively analyze hES cells after 6 passages of LD-1 culture by using flow cytometry.

[0097] 2. Materials hES cells from the first, second and third groups, anti-Oct4, etc.

[0098] 3. Methods Digest with trypsin / EDTA and resuspend in PBS (containing 10% goat serum) at 4°C for 15 minutes. After adding anti-Oct4 monoclonal antibody, incubate at 4°C for 30 min, and then add FITC fluorescein-labeled secondary antibody. The upper flow cytometer (Beckman Coulter Epics XL) was detected, and the data was analyzed by Coulter SYSTEM II software.

[0099] 4. Results The Oct4-positive rate of hES cells in the first and second groups was over 85%; the Oct4-positive rate of hES cells in the third group was less than 20%

[0100] V. Conclusion The hES cells in the first and second groups can basically maintain their undifferentiated state.

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PUM

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Abstract

A human embryonic stem cell culture medium LD-1 independent to feeder cell (fibroblast or others) features that it contains some growth factor, such as fibre growth factor 2 (bFGF) and Noggin, which can support the self updating of human embryonic stem cells.

Description

Technical field: [0001] The present invention relates to a culture medium for human embryonic stem cells (LD1 cell culture medium) that does not depend on feeder cells, in particular to adding some factors that support the self-renewal of human embryonic stem cells to ordinary human embryonic stem cell culture medium, and does not depend on mouse embryogenesis Human embryonic stem cell culture medium for fibroblasts or any other cells. Background technique: [0002] In 1998, Professor Thomson of the University of Wisconsin in the United States successfully isolated the most primitive undifferentiated inner cell mass (Inner Cell Mass, ICM) in early human embryos (blastocysts 5-7 days after fertilization) and inoculated them into monolayer cultures. Mouse embryonic fibroblasts (MEFs) were continuously cultured and passaged in vitro, and kept in an undifferentiated state, and they were called human embryonic stem cells (hereinafter referred to as human embryonic stem cells or h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08
Inventor 李东升邓宏魁
Owner SHIYAN TAIHE HOSPITAL
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