Culture method for colorectal cancer organoids and culture solution

An organ culture, colorectal cancer technology, applied in the culture process, tissue culture, 3D culture and other directions, can solve the problems of lack of widespread use, technical difficulty, complicated operation, etc., to reduce the tedious operation, reduce the risk of pollution, operation easy effect

Active Publication Date: 2021-01-26
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this technology is not widely used due to high cost, complicated operation and technical difficulty.

Method used

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  • Culture method for colorectal cancer organoids and culture solution
  • Culture method for colorectal cancer organoids and culture solution
  • Culture method for colorectal cancer organoids and culture solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] This embodiment provides L-WRN CRL-3276 TM The preparation of the conditioned medium of the cells self-produced Wnt-3a, R-Spondin1 and Noggin three cytokines, including:

[0075] 1) Thaw low-passage L-WRN cells, culture them in conventional DMEM medium containing 10% FBS for one day, and then replace with conventional medium adding G418 (500 μg / mL) and hygromycin (500 μg / mL) until confluence.

[0076] 2) Digest with TE at 37°C for 3-5 minutes, resuspend with regular culture medium, and passage ratio is 1 / 5.

[0077] 3) After culturing for 3-4 days until confluence, the medium was replaced with primary medium (Advanced DMEM / F12+20% FBS+2mM GlutaMAX+1% double antibody).

[0078] 4) After culturing for 24 hours, collect the supernatant for the first time, and centrifuge at 2000g for 5 minutes. After collecting the supernatant, continue culturing with the same volume of primary culture medium, and collect the supernatant every 24 hours. The supernatants of the second, thi...

Embodiment 2

[0081] 1. In vitro processing of colorectal cancer biopsy tissue

[0082] 1) The tumor tissue (about 0.5 g) of the protruding solid part of the top of the colorectal cancer tumor was excised at multiple points. Cryomedium is preserved for delivery to the laboratory. The inventors found in multiple cultures that when taking about 0.5 g of tumor tissue / block, a sufficient amount of single cells can be obtained without wasting a large amount of digestive juice.

[0083] 2) Wash with ice-cold 1xPBS containing antibiotics (1% double antibody (penicillin-streptomycin) + 100ug / ml primocin + 50ug / ml getamicin) up and down for about ten times until the supernatant is clear.

[0084] 3) Place the tissue on ice and shear until there are no visible particles.

[0085] The digestion solution was made into 1mg / mL collagenase IV, 100ug / mL DNAse, and 100ug / ml hyaluronidase digestion working solution. Filter through a 0.22um sterile filter and preheat to 37°C before use. Add 10mL of digest...

Embodiment 3

[0095] Cryopreservation of colorectal cancer organoids

[0096] 1) Collect organoids grown after five days of growth within a certain generation, add cell recovery solution and place on ice for 30 minutes to melt Matrigel.

[0097] 2) Centrifuge, discard the supernatant, resuspend the pellet with 500 μL of Tryple, place it at 37° C. for 3 minutes, and add culture medium to stop the digestion.

[0098] 3) Centrifuge again and discard the supernatant. According to the amount of organoids in one well of a six-well plate, add 1 mL of cryopreservation solution for cryopreservation, about 500,000 organoid fragments / 1 mL of cryopreservation solution, and the component of the cryopreservation solution is FBS containing 10% DMSO. Cryopreservation uses a programmed cryopreservation gradient cooling method.

[0099] see results Figure 7 ,From Figure 7 It can be seen that organoids can still grow stably after cryopreservation and rethawing without affecting cell viability.

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Abstract

The invention relates to a culture method for colorectal cancer organoids and a culture solution. Culture media use cell lines producing Wnt-3a, R-Spondin1 and Noggin to replace the addition of threeexpensive cytokines, so that the costs of culturing organoids can be saved to a utmost extent compared with the formulas of organoid culture media in literature, and success rate can reach more than 90%. The culture solution can culture the tumor tissue derived from the colorectum of patients. The culture method is simple in operation; passage can be realized in five days; solutions do not need tobe changed frequently within five days; and tedious operation can be reduced, and the costs can be saved.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, and in particular relates to a culture method and culture solution for colorectal cancer organoids. Background technique [0002] A major challenge in cancer treatment is to determine whether a localized disease will progress to a malignant phenotype and how to best "match" treatment according to the type and stage of the disease. It is now widely accepted that for many cancers they are no longer a single type of disease but rather a group of cancer types that respond differently to drugs based on histopathological classification. For many years, basic cancer research and anticancer drug development have utilized primary cancer cells and in vitro cultured cell lines grown in tissue culture dishes. While this approach has yielded many of the currently used anticancer drugs, recent studies have found that this approach works poorly at mimicking tumor growth in vivo. In most cases, two-dimens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N5/0679C12N2501/415C12N2501/11C12N2500/60C12N2513/00C12N2501/155C12N2501/727C12N2501/345C12N2500/32C12N2500/38C12N2501/02
Inventor 王均甘丽琴陈安娜沈松陈转鹏梁洁唐世帆
Owner SOUTH CHINA UNIV OF TECH
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