A kind of urea-derived bladder cancer cell culture medium and in vitro culture method of urea-derived bladder cancer cells
A bladder cancer cell culture technology in vitro, which is applied in the field of urinary bladder cancer cell culture medium and urine-derived bladder cancer cell culture in vitro, and can solve the problems of cumbersome operation of bladder cancer cells, unsustainable subculture, easy failure cells, etc. problem, to achieve the effect of solving culture failure, increasing cell number and activity, and eliminating cell contamination
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Embodiment 1
[0054] [Example 1] Urine sampling:
[0055] (1) Sampling objects: male patients with bladder cancer;
[0056] (2) Sampling requirements: Before urine collection, strenuous exercise such as running and climbing stairs should be avoided, and contamination by various substances such as semen, prostatic fluid, and feces should be avoided (urine samples should be sampled before surgery for bladder cancer patients);
[0057] (3) Preparation before the experiment: add 20mL of sample collection solution into a 50mL sterile centrifuge tube to collect urine;
[0058] (4) Clean the urethral area with tincture of benzalkonium bromide, discard the first section of urine, take the interrupted urine, and collect it into the above-mentioned centrifuge tube, each tube can collect 20mL of urine, each patient collects 3 tubes, refrigerated transported to the cell culture room.
[0059] According to the above method (such as figure 1 ) to collect urine samples for primary and subculture of uri...
Embodiment 2
[0060] [Example 2] Primary culture of urethrogenic bladder cancer cells, changing medium:
[0061] (1) The sample was centrifuged at 1500rmp for 8min, and the supernatant was discarded by centrifugation;
[0062] (2) Add 10 mL of PBS buffer solution to wash twice, centrifuge at 1000 rpm for 5 min, and discard the supernatant;
[0063] (3) Resuspend the pellet with 1mL of BLM medium, and inoculate it in a T25 culture bottle, supplement the medium to 7mL in each bottle, and add cells that lose their proliferative ability but still maintain metabolic activity after radiation irradiation or drug mitogenin C treatment. Mouse fibroblasts are about 2 x 10 6 cells / mL co-culture, put in 37 ℃, 5% CO 2 After culturing in the incubator for 36 hours, observe the growth status of the cells under a microscope. The mouse fibroblasts are the mouse fibroblasts MFC / HL-041 in the application number: 201810335862.8 Chinese patent application, which are preserved in the China Center for Type Cult...
Embodiment 3
[0067] [Example 3] Subculture of urogenous bladder cancer cells:
[0068] (1) When the fusion degree of the above-mentioned urinary bladder cancer cells reaches more than 85%, it can be digested and passaged, and the old culture medium is discarded;
[0069] (2) Rinse the cells with PBS buffer to remove cell secretions that hinder trypsin digestion, wash twice, and the operation steps must be gentle;
[0070] (3) After adding 1 mL of EDTA with a concentration of 0.02%, shake it gently to make it fully contact with the cells on the culture flask, and tap the outer wall of the culture flask after 20-30 seconds to make the mouse fibroblasts fall off the wall of the culture flask. After the mouse fibroblasts were all detached from the bottle wall under the microscope, EDTA was quickly removed, and the bladder cancer cells were washed with PBS for 3 times;
[0071] (4) Add 1mL 0.05% trypsin-EDTA, incubate at 37°C for 4min to digest, shake the cell culture flask lightly to make the...
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