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
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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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