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Culture medium and method for establishing pancreas or pancreatic cancer organoid and application

A technology for pancreatic cancer and organoids, which is applied in the field of establishing culture medium for pancreatic or pancreatic cancer organoids, can solve the problems of incurable patients with advanced stage, there is no individualized treatment plan and selection for patients, and achieves the ability to accelerate proliferation and self-assembly Effect

Pending Publication Date: 2022-05-03
重庆嘉士腾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For patients with locally advanced or metastatic disease, standard treatment options are combined chemotherapy regimens. Unfortunately, these cytotoxic drugs do not cure the vast majority of patients with advanced disease, and the median overall survival of these patients is less than one year. In addition, long-term systemic Chemotherapy is associated with toxicity in patients, leading to challenges for patients maintaining these regimens
In addition, many patients have chemotherapy-refractory disease for which there are currently no individualized treatment options

Method used

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  • Culture medium and method for establishing pancreas or pancreatic cancer organoid and application
  • Culture medium and method for establishing pancreas or pancreatic cancer organoid and application
  • Culture medium and method for establishing pancreas or pancreatic cancer organoid and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Establishment of culture medium for pancreas or pancreatic cancer organoids

[0034] Add each component to DMEM / F12 medium at the following concentrations to obtain medium A for culturing pancreas or pancreatic cancer organoids:

[0035]Formula 1: Estradiol 100nM, Hydrocortisone 50μg / ml, R-Spondin 3 (250ng / ml), Neuregulin1 (5nM), FGF10 (50ng / ml), EGF (5ng / ml), PGE2 (5ng / ml ), Noggin (100ng / ml), A83-01 (TGF-β type I receptor inhibitor, 500nM), Wnt3a (5nM), Y-27632 (protein kinase p160ROCK inhibitor, 5μM), SB202190 (p38MAPK inhibitor, 500ng ), B27 (10ng / ml), N-acetylcysteine ​​(N-Acetylcysteine, 5nM), Nicotinamide (Nicotinamide, 10nM), GlutaMax supplement (5nM), fetal bovine serum (FBS, 10%), green chain Penicillin / Streptomycin (1%).

[0036] Formula 2: Estradiol 10nM, Hydrocortisone 0.1μg / ml, R-Spondin 3 (100ng / ml), Neuregulin1 (1nM), FGF10 (10ng / ml), EGF (1ng / ml), PGE2 (1ng / ml ml), Noggin(50ng / ml), A83-01(100nM), Wnt3a(1nM), Y-27632(1μM), SB202190(100ng), ...

Embodiment 2

[0039] Example 2. Method for Establishing Pancreatic Cancer Organoids

[0040] In this example, the samples of pancreas and pancreatic cancer were all from Chongqing People's Hospital.

[0041] The method for establishing pancreatic cancer organoids using the culture medium of Example 1, the specific steps are as follows:

[0042] 1. Pancreatic cancer tissue acquisition

[0043] After collection, the obtained pancreatic cancer tissue / pancreatic tissue was soaked in 50 ml of DMEM / F12+FBS (10%)+Penicillin / Streptomycin (1%).

[0044] 2. Culture of Pancreatic Cancer Organoids

[0045] (1) Soak the obtained pancreatic cancer sample in 1% Penicillin / Streptomycin (penicillin / streptomycin) for 1 hour, take out the soaked pancreatic cancer sample, and chop the cells with a disposable scalpel. The chopped tissue is about 1cm×1cm×1cm; a 1cm×1cm×1cm pancreatic cancer surgical sample was crushed with a grinding rod to prepare a single cell suspension.

[0046] (2) Aspirate the single c...

Embodiment 3

[0048] Example 3. Detection of KRAS G12C Gene Copy Number in Pancreatic Cancer Organoids

[0049] 1. Total RNA extraction

[0050] 1. Weigh quantitative normal pancreatic tissue and pancreatic cancer organoid samples in a pre-cooled mortar, add liquid nitrogen to thoroughly grind them into powder, transfer them to a clean EP tube, add Trizol reagent (1ml / 100mg tissue), and use * Blow and suck repeatedly until there is no obvious precipitation in the lysate, and let stand at room temperature for 5 minutes;

[0051] 2. Add chloroform (0.2ml / ml Trizol), shake the EP tube vigorously for 15 seconds, and let stand at room temperature for 3 minutes;

[0052] 3. Centrifuge at 12,000 rpm for 15 minutes at 4°C and divide into three layers. RNA is in the upper aqueous layer, and the volume of the aqueous layer is about 60% of the amount of Trizol used; the protein exists in the lower organic layer, which can be extracted with isopropanol and stored at 4°C. refrigerator;

[0053] 4. Ab...

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PUM

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Abstract

The invention discloses a culture medium and method for establishing pancreas or pancreatic cancer organoid and application, and the culture medium for establishing the pancreas or pancreatic cancer organoid can rapidly culture the pancreatic cancer organoid and form a 3D pancreatic cancer organoid which is highly consistent with pancreatic cancer tissue in genotype and morphology by blending components of the culture medium. The culture medium is a DMEM / F12 complete culture medium containing the following components: estradiol, hydrocortisone, R-Spondin 3, Neuregulin 1, FGF10, EGF (Epidermal Growth Factor), PGE2, Noggin, A83-01, Wnt3a, Y-27632, SB202190, B27, N-acetylcysteine, nicotinamide, GlutaMax, fetal calf serum and penicillin / streptomycin double antibodies, and the identification method comprises the combination of KRAS G12C gene copy number detection and morphological identification.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a culture medium, method and application for establishing pancreas or pancreatic cancer organoids. Background technique [0002] Despite advances in cancer treatment in recent years, pancreatic cancer still has the lowest survival rate among common malignancies. Late diagnosis and lack of treatment options are the most pressing clinical problems for this disease. Therefore, clinical application of patient models and biomarkers is needed to determine the most effective treatments for patients. Pancreatic organoids are in vitro models of PDAC that can be established from very small biopsies, enabling studies in localized, advanced and metastatic patients. Organoid models have been used in the study of pancreatic cancer, providing a promising platform for precision medicine. Pancreatic ductal adenocarcinoma remains one of the deadliest and most aggressive types of cancer, wit...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09C12Q1/6886C12Q1/02
CPCC12N5/0677C12N5/0693C12Q1/6886G01N33/5076G01N33/507C12N2501/392C12N2501/39C12N2501/998C12N2501/13C12N2501/119C12N2501/11C12N2501/02C12N2501/415C12N2501/727C12N2501/15C12N2500/32C12N2500/38C12Q2600/136G01N2500/10
Inventor 黄璘聂盼万源
Owner 重庆嘉士腾生物科技有限公司
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