Small technique - the primary culture of fibroblasts
Fibroblasts can provide the growth environment needed for embryonic stem cells and maintain an undifferentiated state.
The commonly used feeding cells are mouse embryonicfibroblasts，MEF or STO fibroblast cell lines.
In addition, considering the animal pollution, can also be user human foreskin fibroblast, HFF (ATCC CRL - 1635, Hs68) or other source of cells (e.g. : the human placental fibroblast), etc., as feeding cells.
The blue part of the picture is of fibroblast nuclei, we know that genetic material "Gene" is contained in the nucleus, and the "Gene" is made up of DNA molecules, when cells receive the order, began to transfer some information to the cell and start some biochemical reaction to get the job done.
The orange part is the cytoskeleton of the fibroblasts.
The establishment of embryonic fibroblasts in mice
1.After mating between 5-6 weeks old female rats and male rats, the female rats with vaginal suppository were bred.
2.After the death of the pregnant female rats on the 13th day after the removal of the cervical spondylosis, the complete uterus was removed and placed in the phosphate buffer solution (PBS).
3.After the death of the pregnant female rats on the 13th day after the removal of the cervical spondylosis, the complete uterus was removed and placed in the phosphate buffer solution (PBS).
4.Put the body into 3 ml injection syringe and repeatedly pass the needle until the fetal body is broken into cell mass.
5.Leave the cell suspension for 10 minutes.
6.Drain upper MEF cells suspension to join including feeding broth medium, at 37 ℃
Culture box (5 % CO2).
Adjust the cell number for a petri dish per milliliter about 10 ^ 5 cells.
7.The next day will not attached with a cell and tissue residue removed, change of new culture, and check the attached and cell proliferation, cell for 2 to 3 full of petri dishes, can conduct successive transfer culture.
8.Successive transfer culture, with PBS (Ca2 + / magnesium 2 + - free) clean 2 times, add 1 ml contains 0.05% trypsin - EDTA solution, in 37 ℃ cultivation in 2 minutes, break the suspension cells, add the MEF medium to suspend the trypsin - EDTA, then suspended cells by 1:3 in plate.
9.Then change the culture medium every two days, and then the cells can reach the above 90% of the time.
The laboratory usually USES a secondary number of 5 to 6 generations for embryonic stem cell culture.