In vitro culture method of colorectal cancer organoid

A colorectal cancer and in vitro culture technology, applied in the field of cell engineering, can solve problems such as low success rate, loss of tumor heterogeneity, limited genetic diversity, etc., achieve high success rate, short time, and improve the effect of culturing rate

Inactive Publication Date: 2018-08-14
王琼 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the long-term culture of cell lines, due to the adaptation to the two-dimensional growth environment, the original physiological characteristics in vivo and the heterogeneity of tumors are lost.

Method used

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  • In vitro culture method of colorectal cancer organoid
  • In vitro culture method of colorectal cancer organoid
  • In vitro culture method of colorectal cancer organoid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] (1) In vitro processing of colorectal cancer biopsy tissue

[0053] Colorectal cancer biopsy tissue was rinsed with 4°C pre-cooled PBS until the supernatant was clear.

[0054] Use a disposable scalpel to cut the tissue into small pieces in a 6cm or 10cm cell culture dish, and the size of the tissue piece is about 5mm.

[0055] Use 20ml of enzymatic digestion solution to transfer the tissue pieces to 50ml Falcon centrifuge tubes. The enzyme digestion solution is composed of: DMEM+2% FBS, 70 units / ml collagen hydrolase, 120 μg / ml neutral protease, 2900 units / ml deoxyribonuclease, 1X penicillin, streptomycin mixed solution.

[0056] Seal the centrifuge tube and incubate it at 37°C for 50 minutes, using a shaker to gently mix the sample during the incubation.

[0057] After the incubation, use a pipette to break up the aggregated tissue clumps, and collect the cells filtered with a 70 μM filter membrane, that is, colorectal cancer single cells.

[0058] Colorectal cance...

Embodiment 2

[0067] (1) In vitro processing of colorectal cancer biopsy tissue

[0068] Colorectal cancer biopsy tissue was rinsed with 4°C pre-cooled PBS until the supernatant was clear.

[0069] Use a disposable scalpel to cut the tissue into small pieces in a 6cm or 10cm cell culture dish, and the size of the tissue piece is about 5mm.

[0070] Use 20ml of enzymatic digestion solution to transfer the tissue pieces to 50ml Falcon centrifuge tubes. The enzyme digestion solution is composed of: DMEM+3% FBS, 80 units / ml collagen hydrolase, 130 μg / ml neutral protease, 3100 units / ml deoxyribonuclease, 1X penicillin, streptomycin mixed solution.

[0071] Seal the centrifuge tube and incubate it at 37°C for 70 minutes, using a shaker to mix the sample gently during the incubation.

[0072] After the incubation, use a pipette to break up the aggregated tissue clumps, and collect the cells filtered with a 70 μM filter membrane, that is, colorectal cancer single cells.

[0073] Colorectal cance...

Embodiment 3

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

[0082] Colorectal cancer biopsy tissue was rinsed with 4°C pre-cooled PBS until the supernatant was clear.

[0083] Use a disposable scalpel to cut the tissue into small pieces in a 6cm or 10cm cell culture dish, and the size of the tissue piece is about 5mm.

[0084] Use 20ml of enzymatic digestion solution to transfer the tissue pieces to 50ml Falcon centrifuge tubes. The enzyme digestion solution is composed of: DMEM+2.5% FBS, 75 units / ml collagen hydrolase, 125 μg / ml neutral protease, 3000 units / ml deoxyribonuclease, 1X penicillin, streptomycin mixed solution.

[0085] Seal the centrifuge tube and incubate it at 37°C for 60 minutes, using a shaker to mix the sample gently during the incubation.

[0086] After the incubation, use a pipette to break up the aggregated tissue clumps, and collect the cells filtered with a 70 μM filter membrane, that is, colorectal cancer single cells.

[0087]Colorectal canc...

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Abstract

The invention discloses an in vitro culture method of a colorectal cancer organoid. The in vitro culture method comprises: 1) pretreatment of colorectal cancer biopsy tissue: washing colorectal cancerbiopsy tissue, shearing, and digesting with enzyme to obtain colorectal cancer single cell; and 2) in vitro culture of the colorectal cancer single cell: mixing the colorectal cancer single cell andmatrigel, solidifying, adding a colorectal cancer organoid culture liquid, and culturing to obtain the colorectal cancer organoid. According to the present invention, the method has characteristics ofsimpleness, good stability and high success rate; and the obtained colorectal cancer organoid has a three-dimensional structure, substantially retains the unique morphological characteristics of thepatient tumor tissue, is highly consistent with the pathological features of the biopsy tissue, and provides the material basis for the establishment of the organoid library and the screening of drugs.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a method for culturing and obtaining organoids from colorectal cancer tissue samples in vitro. Background technique [0002] Colorectal cancer is one of the main malignant tumors in humans, threatening human health. The study of its pathogenesis found that the abnormality of WNT, RAS-MAPK, PI3K, TP53, TGF and other signaling pathways plays a key role in the occurrence of colorectal cancer. In addition, with the further in-depth research on colorectal cancer genome, it is found that patients with colorectal cancer have microsatellite DNA instability or chromosomal instability. Compared with other cancers, colorectal cancer has more genetic abnormalities and diversity, and patients often Having multiple gene mutations or multiple gene abnormalities. Therefore, it is impossible to evaluate the general efficacy of the treatment plan for patients only through the analysis of limited ...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2501/11C12N2501/113C12N2501/115C12N2501/117C12N2501/119C12N2501/155C12N2501/415C12N2501/727
Inventor 王琼李曦
Owner 王琼
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