Primary culture method for maintaining near term growth of large intestinal cancer tumour cell
A technique for tumor cell, primary culture
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Embodiment 1
[0034] (1) Obtain fresh tumor specimens in the operating room; wash with sterile saline and cut off the dirty and necrotic areas, soak in iodine and shake gently for 5 minutes; wash with PBS containing double antibodies (1000U / ml penicillin and streptomycin) Wash twice with liquid; transfer to a clean glass dish after disinfection.
[0035] (2) Use sterile ophthalmic scissors to cut the tissue block into as small as possible (<1mm); put it in a 15ml centrifuge tube and let it settle, remove the supernatant, then wash it again with PBS cleaning solution, centrifuge and remove the supernatant .
[0036] (3) Transfer to a 50ml centrifuge tube, add about 10-15ml of collagenase digestion solution (collagenase 1mg / ml + hyaluronidase 0.5mg / ml) according to the total amount of tissue pieces at a ratio of 1:10, and place in a 37°C water bath shaker Neutralization takes 0.5-3 hours, and the specific time can be 0.5 hours, 1 hour, 2 hours or 3 hours according to the actual degree of dig...
Embodiment 2
[0056] (1) Obtain fresh tumor specimens in the operating room; wash with sterile saline and cut off the dirty and necrotic areas, soak in iodophor for 5 minutes for disinfection; wash twice with PBS containing double antibodies (1000U / ml penicillin and streptomycin) ; Transfer to a clean glass dish after disinfection.
[0057] (2) Use sterile ophthalmic scissors to cut the tissue block into fine tissue blocks (average 1--2 mm) (not too fine, otherwise the tissue is easy to float).
[0058] (3) Centrifuge in a 15ml centrifuge tube; discard the supernatant with a dropper, resuspend with PBS cleaning solution and rinse again, centrifuge, discard the supernatant; then add serum-free DMEM culture medium to resuspend, wash , centrifuged, and the supernatant was discarded.
[0059] (4) Add an appropriate amount of collagenase digestion solution (collagenase 1mg / ml + hyaluronidase 0.5mg / ml) according to the amount of the tissue block, and place it in a water bath shaker at 37°C for 3...
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