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Primary culture method for maintaining near term growth of large intestinal cancer tumour cell
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A technique for tumor cell, primary culture
Inactive Publication Date: 2011-07-06
THE AFFILIATED SIR RUN RUN SHAW HOSPITAL OF SCHOOL OF MEDICINE ZHEJIANG UNIV
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Benefits of technology
This patented technology allows culturing small colonic lymphocytes that are resistant against chemotherapy drugs better at treating metastatic breast carcinoma patients who have had their disease over time due to poor outcomes from traditional treatment strategies such as surgery or radiation therapies. By growing these tiny cell populations into larger tissue structures called organoids (organs containing multiple types of immune system components), they could be used experimentally to study how different chemicals might work together specifically towards developing effective therapeutic agents targeted toward specific molecular targets associated with certain diseases like lung cancer.
Problems solved by technology
This patented technical problem addressed in this patents relates to finding an effective way to grow isolated and pure cultured colonic carcinoma (CRC) cells during their own residence inside our body without causing any harmful side effects like those caused by other organisms such as germs or viruses.
Method used
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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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Abstract
The invention pertains to the primary culture technical field of colorectal cancer tumor cells. The method includes: 1) obtaining a fresh tumor sample by operation, cleaning and cutting the pollution and necrosis areas, immersing in iodophor for disinfection, adopting a PBS cleaning liquid containing dual-antigen for cleaning; 2) cutting the tumor sample into small tissue blocks, still placing, removing the supernatant liquid, using the PBS cleaning liquid containing dual-antigen for cleaning, carrying out centrifugal separation and removing the supernatant liquid; 3) adopting collagenase digestion method or tissue block adhering wall method to carry out the tissue adhering wall and cell climbing out in a culture liquid; 4) continually culturing and carrying out purification processing inthe culture liquid to maintain the necessary number for the cell growth. The success rate of the conventional primary culture method for culturing the colorectal cancer tumor cells with good state isvery low (6 percent), however, the primary culture method of the invention can achieve more than 33 percent, thus significantly improving the success probability of short-term in vitro culture of theprimary colorectal cancer cells; furthermore, the technique is simple and easy, the cost is low, so as to provide a good optional platform for applying the experimental studies of the short-term primary colorectal cancer cells.
Description
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Claims
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Application Information
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Owner THE AFFILIATED SIR RUN RUN SHAW HOSPITAL OF SCHOOL OF MEDICINE ZHEJIANG UNIV