Culture method for screening tumor organoids based on P53 mutation

A culture method and organoid technology, applied in the field of culture of tumor organoids based on P53 mutation screening, can solve the problems of inability to detect P53 gene mutation in advance, high cost of gene sequencing, complicated operation, etc., and achieve obvious cost-price advantages and culture results. Stable, highly sensitive effect

Pending Publication Date: 2021-07-30
南昌五元生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cells with p53 deficiency do not have this control and continue to divide even under unfavorable conditions
[0006] The main basis for selecting targeted drugs in clinical research and treatment is tumor gene sequencing. Although tumor organoid culture methods can accurately simulate the tumor microenvironment in vivo and obtain organoid models after stable passage, they cannot detect P53 gene mutations in advance. Therefore, genetic sequencing is needed to determine whether there is a P53 mutation in organoids
However, gene sequencing is expensive, complicated to operate, and requires a large sample size

Method used

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  • Culture method for screening tumor organoids based on P53 mutation
  • Culture method for screening tumor organoids based on P53 mutation
  • Culture method for screening tumor organoids based on P53 mutation

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Effect test

Embodiment 1

[0051] A culture method for screening tumor organoids based on P53 mutation, comprising the following steps:

[0052] S1. Preheat the 24-well plate, basal medium, PBS buffer, and organoid medium containing y27632 in advance, pre-cool the Matrigel matrix in advance, and sterilize the consumables required for the experiment by UV for 30 minutes in advance;

[0053] S2. Dispense 10ml of basal culture into a 15ml centrifuge tube, thaw untreated tumor organoids in a 37°C water bath to generate a cell suspension, add 0.5ml of the cell suspension to the above 10ml of basal medium, and mix by pipetting ;Centrifuge the centrifuge tube at 0°C and 333g for 5 minutes, remove the supernatant, and obtain the tumor organoid mixture;

[0054] S3. Add matrigel to resuspend the tumor organoid mixture, inoculate 40 μl of cell suspension per well into a 24-well plate; after the matrigel solidifies, add 500 μl of preheated organoids containing y27632 (molar concentration: 10 μM) to each well for c...

Embodiment 2

[0057] A culture method for screening tumor organoids based on P53 mutation, comprising the following steps:

[0058] S1. Preheat the 24-well plate, basal medium, PBS buffer, and organoid medium containing y27632 in advance, pre-cool the Matrigel matrix in advance, and sterilize the consumables required for the experiment by UV for 30 minutes in advance;

[0059] S2. Dispense 10ml of basal culture into a 15ml centrifuge tube, thaw untreated tumor organoids in a 37°C water bath to generate a cell suspension, add 0.5ml of the cell suspension to the above 10ml of basal medium, and mix by pipetting ;Centrifuge the centrifuge tube at 0°C and 333g for 5 minutes, remove the supernatant, and obtain the tumor organoid mixture;

[0060] S3. Add matrigel to resuspend the tumor organoid mixture, inoculate 40 μl of cell suspension per well into a 24-well plate; after the matrigel solidifies, add 500 μl of preheated organoids containing y27632 (molar concentration: 10 μM) to each well for c...

Embodiment 3

[0063] A culture method for screening tumor organoids based on P53 mutation, comprising the following steps:

[0064] S1. Preheat the 24-well plate, basal medium, PBS buffer, and organoid medium containing y27632 in advance, pre-cool the Matrigel matrix in advance, and sterilize the consumables required for the experiment by UV for 30 minutes in advance;

[0065] S2. Dispense 10ml of basal culture into a 15ml centrifuge tube, thaw untreated tumor organoids in a 37°C water bath to generate a cell suspension, add 0.5ml of the cell suspension to the above 10ml of basal medium, and mix by pipetting ;Centrifuge the centrifuge tube at 0°C and 333g for 5 minutes, remove the supernatant, and obtain the tumor organoid mixture;

[0066] S3. Add matrigel to resuspend the tumor organoid mixture, inoculate 40 μl of cell suspension per well into a 24-well plate; after the matrigel solidifies, add 500 μl of preheated organoids containing y27632 (molar concentration: 10 μM) to each well for c...

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Abstract

The invention relates to the technical field of organoid culture, and particularly discloses a culture method for screening tumor organoids based on P53 mutation. The culture method comprises the following steps: S1, unfreezing untreated tumor organoid to generate a cell suspension, adding the cell suspension into a basic culture medium, uniformly conducting mixing and centrifuging, and removing supernate to obtain a tumor organoid mixture; S2, adding matrigel to resuspend the tumor organoid mixture, and after resuspending, inoculating the organoid mixture into an organoid culture medium containing y27632 for culturing; and S3, after the liquid of the tumor organoid mixture is changed for the first time, absorbing and removing the organoid culture medium, and then adding a culture medium containing Nutlin3A for continuous culture. The culture method is low in cost and price, sequencing is not needed, the detection cost is saved in the early stage of culture of the tumor organoids, and accurate and simplified culture of the tumor organoids is achieved.

Description

technical field [0001] The invention relates to the technical field of organoid culture, in particular to a method for culturing tumor organoids based on P53 mutation screening. Background technique [0002] At present, with the continuous deepening of research on targeted drugs, a variety of drugs targeting specific oncogenes have entered the clinic, bringing good news to the majority of patients. However, since the essence of cancer is a disease affected by multiple gene mutations, patient tumors will continue to evolve as the disease progresses, resulting in tumor heterogeneity between patients and heterogeneity within the same tumor. Cancer heterogeneity adds uncertainty to targeted precision therapy, and is also one of the important reasons leading to resistance to cancer drug therapy. Therefore, it is of great practical significance to expand precision medicine for cancer heterogeneity, and the rise of tumor organoid technology provides a new and more effective applic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2501/06C12N2501/727
Inventor 杨光周正牛建军刘平果张以平
Owner 南昌五元生物科技有限公司
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