1. Separation method of suspension cells
1) The suspension of blood, amniotic fluid, pleural fluid or ascites was directly transferred into a centrifuge tube and centrifuged at 1000 rpm / min for 5 minutes.
2) The supernatant was removed, centrifuged and precipitated with PBS without calcium or magnesium, and centrifuged at 1000 rpm / min for 5 minutes. Repeat this step twice.
3) The cells were resuspended with medium and cultured in bottles after adjusting the appropriate cell concentration.
4) If a certain cell is selected in the suspension, the centrifuged cell layering solution can be used to harvest the target cells.
2. Separation method of solid tissue materials
1) Mechanical dispersion method
a. Brain tissue with little fiber composition and part of embryonic tissue were cut to 1mm3 tissue block with scissors.
b. After cleaning twice with PBS
① Blow with a straw to disperse tissue cells.
② Or put the fully cut and scattered tissue into the syringe (with No. 9 needle) to press out the cells through the needle.
③ Or in stainless steel gauze with blunt object (blunt end of syringe) to squeeze cells from mesh.
c. The cells were collected and transferred into a centrifuge tube and centrifuged at 1000 rpm / min for 5 minutes.
d. The supernatant was removed, and the culture medium containing serum was added. After suspension, it was transferred to the culture bottle for culture.
2) Digestion separation method
Enzymatic digestion and separation (overnight cold digestion)
a. Soft tissue with less intercellular substance, such as liver, kidney, thyroid, amnion, embryonic tissue and epithelial tissue, should be washed with Hanks solution for three times and cut into pieces about 4 mm in size.
b. Wash 2-3 times with Hanks solution to remove blood cells and adipose tissue.
c. Add 0.25% trypsin, shake well and put it at 4 ℃ overnight.
d. The next day, wash with Hanks solution, discard the supernatant, and wash 2-3 times.
e. Add a small amount of serum containing medium, blow and disperse, count cells, and culture in bottles according to appropriate concentration.
Non enzymatic digestion (EDTA digestion)
a. The tissue block was cut into pieces, and the tissue mass was 1mm3 in size.
b. Wash the broken tissue block in the plate with PBS without calcium and magnesium for 2-3 times.
c. Add the digestive solution (trypsin or collagenase or EDTA) into the water bath at 37 ℃ for a proper time (gently shaking 1-2 times in the middle). If the tissue block is bulky and flocculent, it can be stopped. If the change is not big, the digestive solution can be replaced once and digestion will continue until it is fluffy.
d. Discard the supernatant, add medium containing calcium and magnesium ions, stop digestion reaction, and wash 2-3 times, then add complete medium.
e. The cells were blown with a pipette, and then the cells were filtered with gauze and cultured in bottles.