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Oral squamous cell carcinoma organoid culture medium and culture method

A technology of oral squamous cell carcinoma and culture method, which is applied in the field of oral squamous cell carcinoma organoid culture medium and culture, can solve the problems of slow growth, high pollution ratio, low culture success rate, etc., and achieve the effect of strengthening tissue pretreatment

Pending Publication Date: 2021-08-20
NANJING STOMATOLOGICAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to provide a culture medium and culture method for oral squamous cell carcinoma organoids, aiming to solve the problems of high pollution ratio, low culture success rate and slow growth in the culture of oral squamous cell carcinoma organoids in the prior art

Method used

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  • Oral squamous cell carcinoma organoid culture medium and culture method
  • Oral squamous cell carcinoma organoid culture medium and culture method

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Experimental program
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Embodiment 1

[0031] An organoid culture medium for oral squamous cell carcinoma, consisting of basal medium DMEM / F12, R-spondin1 conditioned medium, Wnt3A conditioned medium, sterile water and functional components, the functional components in the organoid medium The final concentration in the composition is: HEPES, 8-12mmol / L; Glutamax 0.8-1.2×; A83-01, 400-600nmol / L; EGF, 35-60ng / mL; Noggin, 80-120ng / mL; FGF10, 8 -12ng / mL; Gastrin I, 0.01μmol / L; N-acetylcysteine, 1-1.5mmol / L; nicotinamide, 8-12mmol / L; PGE2, 0.8-1.2μmol / L; Butylatedhydroxyanisole, 4-10ng / ml; Penicillin - Streptomycin-Amphotericin B mixed solution 0.8-1.2X; B27supplement; Prostaglandin E2, 0.8-1.2umol / L; and R-spondin1 conditioned medium accounted for 10% of the total volume, Wnt3A conditioned medium accounted for 10% of the total volume 50%.

Embodiment 2

[0033] An organoid culture medium for oral squamous cell carcinoma, consisting of basal medium DMEM / F12, R-spondin1 conditioned medium, Wnt3A conditioned medium, sterile water and functional components, the functional components in the organoid medium The final concentration in the composition is: HEPES, 10mmol / L; Glutamax 1×; A83-01, 500nmol / L; EGF, 50ng / mL; Noggin, 100ng / mL; FGF10, 10ng / mL; Gastrin I, 0.01μmol / L ; N-acetylcysteine, 1.25mmol / L; nicotinamide, 10mmol / L; PGE2, 1μmol / L; Butylated hydroxyanisole, 5ng / ml; penicillin-streptomycin-amphotericin B mixed solution 1X; B27 supplement; Prostaglandin E2, 1umol / L; and the R-spondin1 conditioned medium accounts for 10% of the total volume, and the Wnt3A conditioned medium accounts for 50% of the total volume.

Embodiment 3

[0035] This embodiment provides a method for culturing oral squamous cell carcinoma organoids, comprising the following steps:

[0036] S1. Dissociative enzyme configuration: 5mg / ml collagenase I and 10ug / ml DNase I were prepared with serum-free DMEM medium as a solvent, and stored at a temperature of 4 degrees;

[0037] S2, configure the culture medium of any embodiment such as embodiment 1 or embodiment 2;

[0038] S3. Obtain samples for clinical operations:

[0039] Obtain tumor specimens by taking materials or surgical resection, cut out 0.5cm×0.5cm×0.5cm tissue, rinse with sterile saline for 3 times, soak the tissue in pre-cooled DMEM medium containing 20% ​​double antibody for 1 hour, Continue to dissociate, and store and transport on ice throughout the process;

[0040] S4. Tissue pretreatment:

[0041] The sample obtained in step S3 is placed in a sterile 6-well plate, and the sample tissue block is chopped with a disposable sterile scalpel;

[0042] S5. Tissue dis...

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Abstract

The invention discloses a culture method of oral squamous cell carcinoma organoid. The culture method comprises the following steps: S1, preparing a dissociation enzyme; S2, preparing a culture medium, wherein the culture medium is composed of a basal culture medium DMEM / F12, an R-spondin1 conditional culture medium, a Wnt3A conditional culture medium, sterile water and functional components; S3, obtaining a sample through clinical operation, namely obtaining a tumor specimen through material taking or surgical resection, cutting tissue, washing the tissue with sterile normal saline for three times, soaking the tissue in a DMEM medium for 1 hour and continuing to dissociate; S4, tissue pretreatment, namely placing the sample obtained in the step S3 in a sterile 6-well plate, and cutting up sample tissue blocks by using a disposable sterile surgical blade; S5, tissue dissociation and digestion, namely adding the dissociation enzyme prepared in the step S1, repeating the process of centrifugation-supernatant removal for three times so as to fully remove the dissociation enzyme, and mixing the cells with the sample tissue blocks before the last time of centrifugation; and S6, carrying out organoid culture.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and more specifically relates to a culture medium and a culture method for oral squamous cell carcinoma organoids. Background technique [0002] Oral squamous cell carcinoma (Oral Squamous Cell Carcinoma, OSCC) is the most common malignant tumor of the oral and maxillofacial region. In 2020, there will be about 377,700 new cases of OSCC in the world, and 177,800 deaths. The 5-year survival rate is only about 60%. For middle-advanced OSCC patients, especially recurrent / metastatic OSCC patients, chemotherapy-assisted comprehensive therapy is a standardized treatment plan. For example, cisplatin and paclitaxel are the first-line chemotherapy drugs, but the therapeutic effect is not satisfactory, and targeted therapy drugs are of great benefit to the patients. The treatment effect of OSCC also needs to be further verified. Therefore, there is an urgent need to develop a more effective individuali...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N5/0632C12N2501/11C12N2501/119C12N2501/02C12N2500/60C12N2501/345C12N2500/32C12N2500/50C12N2500/30C12N2533/90
Inventor 章茜王育新胡勤刚
Owner NANJING STOMATOLOGICAL HOSPITAL
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