Methods for culturing and identifying nasopharyngeal carcinoma organs

Abstract

The application relates to a culture medium for nasopharyngeal carcinoma organs, and methods for culturing and identifying the nasopharyngeal carcinoma organs. According to the culture and growth characteristics of nasopharyngeal carcinoma-derived cells, a variety of cytokine components are selected and blended according to a certain ratio; the contents of cytokines and signal pathway regulatory factors in the culture medium are suitable; the nasopharyngeal carcinoma cells can effectively form organoids in a three-dimensional (3D) environment. The methods for culturing and identifying the nasopharyngeal carcinoma organs as well as the unique culture medium for the nasopharyngeal carcinoma organs can be used for successfully and stably culturing the nasopharyngeal carcinoma organs which have pathophysiological features and genotypes being highly consistent with those of the parental tumor and have high similar form of organization.

Description

technical field

[0001] The invention relates to the field of organoid culture, and further relates to the field of nasopharyngeal carcinoma organoid culture, in particular to a culture medium, a culture method and an identification method for nasopharyngeal carcinoma organoids. Background technique

[0002] In my country, nasopharyngeal carcinoma has become the main cancer and cause of death in malignant tumors of the ear, nose, and throat, especially in Guangdong and Guangxi in southern China (such as Guangdong, Guangxi, etc.). Radiation therapy is the first choice for nasopharyngeal carcinoma. Radiotherapy alone for stage Ⅰ and Ⅱ nasopharyngeal carcinoma can achieve a good therapeutic effect, and the 5-year overall survival rate of initial treatment can reach 75%-84.5%. However, the rate of recurrence and metastasis after the first course of treatment is as high as 10-36%. In addition, the effect of re-radiotherapy and chemotherapy is not good for patients with recurrent ...

Examples

Embodiment 1

[0046] A culture medium for nasopharyngeal carcinoma organoids, the medium comprising the following components: B27, 50X dilution; N-acetylcysteine ​​1mM; EGF 5ng / ml; Noggin 100ng / ml, R-spondin 1 250ng / ml; A83-01500nM ; HGF 10ng / ml; Nicotinamide 10mM; Y-27632 10μM; Wnt3a 250ng / ml; Glutmax 100X dilution; Heregulinβ-1 20ng / ml; 10ng / ml.

Embodiment 2

[0048] According to the method for culturing nasopharyngeal carcinoma organoids in the culture medium of Example 1, the specific method is as follows:

[0049] Shred the tumor tissue on ice, add organoid digestive enzyme I to resuspend, transfer to 37°C, 220rpm constant temperature shaker for digestion for about 1.5 hours; after the first digestion, centrifuge to discard the supernatant, add 2ml digestive enzyme II to pipette Resuspend, place the centrifuge tube in a 37°C, 220rpm constant temperature shaker for 10 minutes, add Hanks solution to stop the digestion, after digestion with digestive enzyme II, single cells are obtained; after the second digestion, discard the supernatant by centrifugation to obtain cell pellets, Add 2ml of Hanks solution to resuspend the digested tissue pellet; filter the cells with a 70μm cell mesh, and centrifuge to discard the supernatant; take 1ml of red blood cell lysate to resuspend the cells for 2-3min, then add 1ml of HBSS to terminate; then...

Embodiment 3

[0052] The organoids cultivated in Example 2 were identified, and the specific identification methods were as follows:

[0053] The organoids were collected, dropped into the OCT embedding medium prepared in advance, frozen at -80°C and sectioned (stained routinely), and the cell type and cell source were identified. Immunofluorescent staining and Q-PCR were used to detect the expression of genes related to nasopharyngeal mucosa tissue derived from cells in the tissue. Organoids, tumor tissues and blood from patients were sent to BGI for full tumor exome and Epstein-Barr virus gene sequencing, which confirmed the homology between tumor tissues and nasopharyngeal carcinoma organoids. Using CRISPR / Cas9 technology to mutate genes in vitro to study the role of related genes in the occurrence and development of nasopharyngeal carcinoma-related diseases.

[0054] The specific steps of homology and stability identification are as follows:

[0055] (1) Whole exome sequencing of naso...

technical field

[0001] The invention relates to the field of organoid culture, and further relates to the field of nasopharyngeal carcinoma organoid culture, in particular to a culture medium, a culture method and an identification method for nasopharyngeal carcinoma organoids. Background technique

[0002] In my country, nasopharyngeal carcinoma has become the main cancer and cause of death in malignant tumors of the ear, nose, and throat, especially in Guangdong and Guangxi in southern China (such as Guangdong, Guangxi, etc.). Radiation therapy is the first choice for nasopharyngeal carcinoma. Radiotherapy alone for stage Ⅰ and Ⅱ nasopharyngeal carcinoma can achieve a good therapeutic effect, and the 5-year overall survival rate of initial treatment can reach 75%-84.5%. However, the rate of recurrence and metastasis after the first course of treatment is as high as 10-36%. In addition, the effect of re-radiotherapy and chemotherapy is not good for patients with recurrent ...

Examples

Embodiment 1

[0046] A culture medium for nasopharyngeal carcinoma organoids, the medium comprising the following components: B27, 50X dilution; N-acetylcysteine ​​1mM; EGF 5ng / ml; Noggin 100ng / ml, R-spondin 1 250ng / ml; A83-01500nM ; HGF 10ng / ml; Nicotinamide 10mM; Y-27632 10μM; Wnt3a 250ng / ml; Glutmax 100X dilution; Heregulinβ-1 20ng / ml; 10ng / ml.

Embodiment 2

[0048] According to the method for culturing nasopharyngeal carcinoma organoids in the culture medium of Example 1, the specific method is as follows:

[0049] Shred the tumor tissue on ice, add organoid digestive enzyme I to resuspend, transfer to 37°C, 220rpm constant temperature shaker for digestion for about 1.5 hours; after the first digestion, centrifuge to discard the supernatant, add 2ml digestive enzyme II to pipette Resuspend, place the centrifuge tube in a 37°C, 220rpm constant temperature shaker for 10 minutes, add Hanks solution to stop the digestion, after digestion with digestive enzyme II, single cells are obtained; after the second digestion, discard the supernatant by centrifugation to obtain cell pellets, Add 2ml of Hanks solution to resuspend the digested tissue pellet; filter the cells with a 70μm cell mesh, and centrifuge to discard the supernatant; take 1ml of red blood cell lysate to resuspend the cells for 2-3min, then add 1ml of HBSS to terminate; then...

Embodiment 3

[0052] The organoids cultivated in Example 2 were identified, and the specific identification methods were as follows:

[0053] The organoids were collected, dropped into the OCT embedding medium prepared in advance, frozen at -80°C and sectioned (stained routinely), and the cell type and cell source were identified. Immunofluorescent staining and Q-PCR were used to detect the expression of genes related to nasopharyngeal mucosa tissue derived from cells in the tissue. Organoids, tumor tissues and blood from patients were sent to BGI for full tumor exome and Epstein-Barr virus gene sequencing, which confirmed the homology between tumor tissues and nasopharyngeal carcinoma organoids. Using CRISPR / Cas9 technology to mutate genes in vitro to study the role of related genes in the occurrence and development of nasopharyngeal carcinoma-related diseases.

[0054] The specific steps of homology and stability identification are as follows:

[0055] (1) Whole exome sequencing of naso...