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In vitro culture method of cancer type organs

An in vitro culture and organ technology, applied in the field of cell engineering, can solve the problems of low enzymatic hydrolysis efficiency, easy contamination of samples, and inability to subculture for a long time, so as to improve enzymatic hydrolysis efficiency and maintain cell viability

Active Publication Date: 2020-01-07
NANOPEPTIDE QINGDAO BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The inventor found that the existing in vitro culture technology of organoids has the problems of easy sample contamination and low enzymatic hydrolysis efficiency, and the existing in vitro culture technology of organoids can only maintain growth in a short period of time, and cannot be subcultured for a long time. Failed to create an organoid biobank

Method used

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  • In vitro culture method of cancer type organs
  • In vitro culture method of cancer type organs
  • In vitro culture method of cancer type organs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] This example aims to explore the best solution for sample digestion, and cut the colon adenocarcinoma sample into 1-2mm 3 Small pieces, randomly divided into 3 parts, corresponding to the following 3 digestion schemes for the above enzymatic digestion process:

[0056] (1) Collagenase type I 75u / mL+Dispase 0.6u / mL;

[0057] (2) Collagenase type I 255u / mL+Dispase 0.6u / mL;

[0058] (3) Collagenase type I 255u / mL.

[0059] After the digestion, cultured according to the above culture process for 3 days. It was found that the solution (2) would cause many vacuoles and apoptosis in the cells, and the solution (3) would lead to a decrease in cell viability, and the dissociated cells did not reunite. , the organoid formation rate decreased, and only scheme (1) obtained better cell viability and could form organoids smoothly. Such as figure 1 shown.

Embodiment 2

[0061] In this plan, the purpose is to explore the best plan for sample cleaning, and 10 samples are selected to be treated with washing buffer in different orders.

[0062] 10 cases in the control group:

[0063] 1) Perform the second washing treatment on the fresh tumor samples in the washing buffer, that is, wash the fresh tumor samples in the washing buffer 1 for 5 or 6 times, each time for 4-6 minutes; Wash in Washing Buffer 2 for 2-4 minutes; wash the tissue washed in Washing Buffer 2 in Washing Buffer 3 for 2-4 minutes; wash the tissue washed in Washing Buffer 3 in Washing Buffer 4 at 4 Soaking for 28-32 minutes under the condition of ℃; and washing the tissue soaked in the washing buffer 4 in the washing buffer 5 for 5 or 6 times, each time for 4-6 minutes.

[0064]2) Excluding the samples after the first cleaning treatment, so as to remove fat, blood, necrosis and tissues with a content of interstitium greater than 20%;

[0065] 3) Perform the first cleaning treatme...

Embodiment 3

[0070] In this protocol, we aim to explore the best protocol for cell culture, and use colon adenocarcinoma organoid samples to investigate the effects of common components of the prior art medium on the growth of organoids.

[0071] The specific protocol is as follows: count the organoids, use Matrigel to resuspend the organoids according to the concentration of 10 μL / 50 organoids, plant them in a 96-well plate, 10 μL / well, and use the culture method minus different single components after seeding the plate. After 5 days of culture, the growth pictures were recorded and the effects of different medium conditions on the growth of organoids were quantitatively detected.

[0072] It was found that complete medium (including 10mM HEPES, 2mM L-Glutamine, 50ng / mLEGF, 100ng / mLNoggin, 500ng / mLLR-Spondin 1, 100ng / mLWNT-3A, 10ng / mL FGF-10, 0.5μM A83-01, 5uMSB202190, 4mM Nicotinamide and 10μM Y27632 in DMEM / F-12K medium), Noggin, A83-01, EGF and Y27632 components can promote the growth ...

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Abstract

The invention provides an in vitro culture method of cancer type organs. According to the embodiment of the invention, the method comprises the steps of performing shearing and digestion treatment oncancer tissue samples, wherein the size of cancer tissue after the shearing treatment is 1-2mm<3>, and the digestion treatment is performed under the action of combined digestive enzymes; and performing re-suspension treatment on cancer cells obtained after digestion treatment with precooling substrate glue, wherein the concentration of the cancer cells in the substrate glue is 500-1500 / 10 [mu]L;and performing point and pallet treatment on cancer cell suspension, and performing culture treatment on the cancer cells after point and pallet treatment is performed, so as to obtain cancer organs.According to the method of the embodiment of the invention, the enzymolysis efficiency of the cancer cells is improved, the success rate of subculturing of organoids is increased, and long-term culture of the organoids can be realized, and a biology sample database can be established.

Description

technical field [0001] The present invention relates to the field of cell engineering, more specifically, the present invention relates to a method for culturing cancerous organoids in vitro. Background technique [0002] Colon adenocarcinoma 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 colon adenocarcinoma. In addition, with the further in-depth study of colon adenocarcinoma genome, it is found that patients with colorectal cancer have microsatellite DNA instability or chromosomal instability. Compared with other cancers, colon adenocarcinoma 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 o...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2509/00C12N2513/00
Inventor 曹志鹏陈璞卓朗
Owner NANOPEPTIDE QINGDAO BIOTECH LTD
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