Culture medium and culture method for nasopharynx cancer organoid

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a culture medium and a culture method for nasopharyngeal carcinoma organs. The culture medium comprises the following components: an antibiotic, a glutamine supplement, a B27 serum-free additive, N-acetylcysteine, nicotinamide, a p38MAPK inhibitor, a TGF-beta I type receptor inhibitor, a recombinant human R type reactive protein, a recombinant human head protein, a recombinant LMP2 expression protein, a ROCK inhibitor, an FGF-10 and an EGF (Epidermal Growth Factor). According to the culture medium, through the combined action of the recombinant human R-type reaction protein, the recombinant human head protein, the recombinant LMP2 expression protein, the ROCK inhibitor, the FGF-10 and the hEGF, the in-vivo microenvironment is simulated, and cell differentiation of nasopharyngeal carcinoma organs is promoted, so that the effects of shortening the culture period and improving the passage frequency and the passage success rate are achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a culture medium and a culture method for nasopharyngeal carcinoma organoids. BACKGROUND

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from nasopharyngeal epithelial cells, and its incidence rate ranks first among ear-nose-throat malignant tumors. NPC is difficult to be found in the early stage of the disease, and is prone to metastasis. Most patients are in the middle and late stages when diagnosed, and have signs of recurrence and metastasis. Due to the regional and rare distribution of NPC, there are few NPC models available for basic and clinical research at present, and the basic and clinical application and transformation research of NPC are relatively backward.

[0003] Organoids are organ-specific cell collections derived from stem cells or precursor cells. Tumor organoids are formed by inducing differentiation of tumor cells with "stemness" through in vitro 3D culture technology. They well retain the heterogeneity and other tumor characteristics of the source tumor tissue in structure and function, and become a new clinical research model for tumor basic research and clinical transformation application. In vitro cultured organoids are highly similar to the corresponding organs in cell composition and tissue architecture, and have corresponding functional characteristics. Unlike conventional cell culture, which cultures single cell groups in a two-dimensional environment, organoid culture is a specific tissue organ cell group that is cultured in a three-dimensional environment. The culture system is more similar to the physiological structure and function of in vivo tissues.

[0004] In recent years, organoid models of various types of tumors have emerged. However, the culture technology and clinical application research of NPC organoids are still in the initial stage. This is mainly due to the great difference between NPC and other tumors in terms of causes and mechanisms, which leads to great differences in organoid culture. The construction of a stable passaged nasopharyngeal carcinoma tumor organoid preclinical research model is of great significance for establishing a preclinical efficacy prediction of nasopharyngeal carcinoma antitumor drugs, guiding the use of drugs in the clinical treatment of nasopharyngeal carcinoma, and promoting individualized precision treatment of nasopharyngeal carcinoma.

[0005] In the related art, the culture medium for nasopharyngeal carcinoma tumor organoids limits the gas exchange and material metabolism of the organoids with the outside world, and when the organoids form a larger tissue, the lack of the circulatory system and the limitation of oxygen nutrient exchange seriously affect the absorption of the required nutrients of the organoids and the removal of metabolic waste, and also limit the subculture of the organoids, resulting in a long subculture period and a low subculture frequency, and the subculture frequency of the existing nasopharyngeal carcinoma organoids is generally about 1-2 generations. In addition, due to the lack of specific in-vivo environment and related immune response, the retention rate of the heterogeneity of the source tumor tissue and other tumor characteristics during the induction and differentiation process is reduced, which also leads to the reduction of the subculture frequency and the reduction of the subculture success rate.

[0006] The organoid immune co-culture technology is an advanced technology for specifically inducing and analyzing tumor immune response by co-culturing the cultured organoids with immune cells to simulate the in-vivo microenvironment and study the interaction between cells. At present, it is mainly used to simulate the in-vivo immune attack of immune cells (such as T cells and NK cells) on tumor cells, but such immune attack is obviously not conducive to the culture formation of organoids. It is of great significance to develop a culture medium and a culture method capable of specifically inducing the culture formation of NPC organoids to shorten the culture period, improve the subculture frequency and the subculture success rate. SUMMARY

[0007] In view of the long culture period, low subculture frequency and low subculture success rate of the nasopharyngeal carcinoma organoids in the prior art, the purpose of the present application is to provide a culture medium and a culture method for nasopharyngeal carcinoma organoids.

[0008] The purpose of the present application is achieved by the following technical solutions. A culture medium for nasopharyngeal carcinoma organoids comprises the following functional components in the following content ranges: antibiotics, 30-120 μmol / mL; glutamine supplement, 15-25 nmol / L; B27 serum-free additive, 0.5%-1.5% by volume fraction; N-acetylcysteine, 0.2-0.3 mg / ml; nicotinamide, 8-12 nmol / L; p38 MAPK inhibitor, 7-12 μmol / mL; TGF-β type I receptor inhibitor, 7-12 nmol / L; recombinant human R-type response protein, 400-6000 ng / mL, recombinant human noggin, 70-130 ng / mL, recombinant LMP2 expression protein, 100-500 ng / mL, ROCK inhibitor, 10-30 μmol / mL; Fibroblast growth factor 10 (FGF-10), 30-60 μmol / mL; Epidermal growth factor (EGF), 30-60 nmo l / mL.

[0009] Preferably, the antibiotic comprises one or more of penicillin, streptomycin, amphotericin B; more preferably a mixture of penicillin, streptomycin and amphotericin B. The antibiotic functions to avoid interference with the development function of the organoid by anti-pathogenic or other active secondary metabolites during multiple subculture.

[0010] Preferably, the p38 MAPK inhibitor comprises one or more of 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole, compound SB203580, compound SB20219, compound SB239063.

[0011] Preferably, the TGF-beta type I receptor inhibitor comprises one or more of compound A83-01, compound SB431542, compound LY2157299.

[0012] Preferably, the amino acid sequence of the recombinant human R-type response protein is shown as SEQ ID NO. 1.

[0013] The recombinant human R-type response protein can be prepared by an E. coli expression system, and the Met1-Leu137 encoded by the target gene contains a His tag at the N terminus. The addition of the recombinant human R-type response protein in the culture medium of the application can shorten the culture period of the nasopharyngeal carcinoma organoid and increase the number of passages.

[0014] Preferably, the amino acid sequence of the recombinant human noggin is shown as SEQ ID NO. 2.

[0015] The addition of the recombinant human R-type response protein in the culture medium of the application can shorten the culture period of the nasopharyngeal carcinoma organoid and increase the number of passages.

[0016] Preferably, the preparation method of the recombinant LMP2 expression protein is as follows: The EB virus latent membrane protein 2 (LMP2) gene (the sequence is shown as SEQ ID NO. 3) is cloned into a pET28a vector to obtain a recombinant plasmid; then the recombinant plasmid is transformed into BL21 (DE3) competent cells for expression to obtain a recombinant LMP2 expression protein.

[0017] LMP2 is one of the viral proteins that is continuously expressed in EB virus-related tumor tissues such as NPC, and the LMP2 gene does not cause transformation of B lymphocytes and is non-carcinogenic. The LMP2 expression protein used in the present application cannot induce specific T cell immune response in the organoid culture system, so it will not cause a killing effect on the culture of the organoid, and will not affect the growth, expansion and survival of the organoid; but the specific induction differentiation effect of the LMP2 expression protein can better simulate the in vivo microenvironment of NPC patients, significantly improve the retention rate of the heterogeneity of NPC tissues and other various oncological characteristics during the induction and differentiation process, thereby achieving the effect of increasing the number of passages and the success rate of passage of the nasopharyngeal carcinoma organoid culture.

[0018] Preferably, the ROCK inhibitor includes one or more of the following: compound Y-27632, compound fasudil, hydroxyfasudil, compound Y-33075, compound T14960, GSK429286A, N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide (Thiazovivin).

[0019] More preferably, the ROCK inhibitor is compound Y-27632.

[0020] Most preferably, the molar ratio of FGF-10, EGF and compound Y27632 is 1: (5x10 -4 -2x10 -3 ):(0.2-1.0).

[0021] A method for culturing a nasopharyngeal carcinoma organoid, comprising the following steps: (1) dispersing an ex vivo active nasopharyngeal carcinoma tissue in a tissue digestion solution and a trypsin solution in sequence for treatment to obtain an active nasopharyngeal carcinoma cell; (2) resuspending the nasopharyngeal carcinoma cell of step (1) with a serum-reduced basal medium, filtering with a molecular sieve and centrifuging to obtain a resuspended nasopharyngeal carcinoma cell by taking the precipitate; (3) resuspending the resuspended nasopharyngeal carcinoma cell of step (2) with a mixture of a serum-reduced basal medium and Matrigel to obtain a target nasopharyngeal carcinoma cell, and then inoculating it into the above-mentioned culture medium for nasopharyngeal carcinoma organoids for culture, and updating the components of the culture medium to the initial state at intervals, to obtain a nasopharyngeal carcinoma organoid.

[0022] Preferably, the tissue digestion solution in step (1) comprises 40-60 mg / ml collagenase type II, 18-22 mg / ml deoxyribonuclease I, 95-105 mg / ml TrypLE Express and 45-55 μg / ml Y-27632; the trypsin solution has a mass concentration of 0.1%-0.25%. Through the above treatment, metabolic waste in the nasopharyngeal carcinoma tissue can be removed, the survival rate can be improved, and deoxyribonucleic acid therein can be removed, so that relatively dispersed single nasopharyngeal carcinoma cells can be obtained.

[0023] Collagenase type II, at a certain concentration, can be used for dissociation of tissues and cells of heart, thyroid, salivary gland, liver, bone and cartilage. In the present application, in combination with other components, dissociation of nasopharyngeal carcinoma tissue with cancer cells is realized.

[0024] Deoxyribonuclease I (DNase I) can be derived from bovine pancreas and is an endonuclease that preferentially hydrolyzes double-stranded or single-stranded DNA from pyrimidine nucleotides.

[0025] TrypLE Express is a non-animal source recombinant enzyme suitable for dissociating various adherent mammalian cells, including CHO, HEK 293, A549, primary human keratinocytes and embryonic stem cells. TrypLE Express can replace pig or bovine trypsin for separating adherent cell lines on plastic products.

[0026] Preferably, in the mixture in step (3), the volume ratio of serum-reduced basal medium (such as Advanced DMEM / F-12) to Matrigel matrix gel (such as Matrigel matrix gel) is 3-10:1.

[0027] Preferably, in step (5), the component of the interval update medium is updated to the initial state at intervals of 3-5 days.

[0028] A nasopharyngeal carcinoma organoid is obtained by the above method.

[0029] The principle of the present application is that, through the activation and regulation of recombinant human R-type response protein and recombinant human protoglobin, the specific induction differentiation of recombinant LMP2 expression protein, and the growth promotion, expansion and survival improvement of ROCK inhibitor, FGF-10 and hEGF, through the combined action of the above soluble factors, the in-vivo microenvironment is simulated, and the cell differentiation of the nasopharyngeal carcinoma organoid is promoted, so that the effects of shortening the culture period, increasing the passage number and improving the passage success rate are achieved.

[0030] Compared with the prior art, the present application has the following beneficial effects: The culture medium of the nasopharyngeal carcinoma organoid of the application is targeted at the growth characteristics of the nasopharyngeal carcinoma tissue-derived cells, and uses the same to realize rapid expansion of tumor cells to form an organoid in a short time. A sufficient number of organoids can be obtained in a short time, for example, a nasopharyngeal carcinoma organoid only needs 1-2 weeks to develop from inoculation to maturation. The components in the culture medium of the application, especially the recombinant human R-type response protein, the recombinant human noggin, the recombinant LMP2 expression protein, the ROCK inhibitor, FGF-10 and hEGF, cooperate with each other to continuously maintain the tissue cell specificity, so that the tissue morphology of the obtained nasopharyngeal carcinoma organoid is also highly similar. Through the culture medium of the application, more than 90% of nasopharyngeal carcinoma samples can successfully form nasopharyngeal carcinoma organoids, and the organoids are highly consistent with the original tissue in histopathology, and can be successfully passaged to 5 generations, and the next passage culture can be carried out every 1-2 weeks. The nasopharyngeal carcinoma organoid obtained using the above culture medium can not only meet the needs of nasopharyngeal carcinoma scientific research, but also provide effective guidance for the clinical treatment of patients by testing the drug sensitivity before the treatment of nasopharyngeal carcinoma patients, and can become a new and effective preclinical in vitro model for precise individualized treatment of nasopharyngeal carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The figure is a schematic diagram of the digestion and inoculation process of the nasopharyngeal carcinoma tissue in Example 1.

[0032] Figure 2 The figure is a variation chart of the culture process of the nasopharyngeal carcinoma organoid in Example 1.

[0033] Figure 3 The figure is a HE staining chart of the nasopharyngeal carcinoma tissue and the cultured nasopharyngeal carcinoma organoid in Example 1.

[0034] Figure 4 The figure is a chart of the immunohistochemical staining results of the nasopharyngeal carcinoma organoid obtained in Example 1.

[0035] Figure 5 The figure is a chart of the immunohistochemical CK5 / 6 detection results of the nasopharyngeal carcinoma tissue and the cultured nasopharyngeal carcinoma organoid in Example 1.

[0036] Figure 6 The figure is a chart of the immunohistochemical P63 detection results of the nasopharyngeal carcinoma tissue and the cultured nasopharyngeal carcinoma organoid in Example 1.

[0037] Figure 7 The figure is a chart of the immunohistochemical P40 detection results of the nasopharyngeal carcinoma tissue and the cultured nasopharyngeal carcinoma organoid in Example 1.

[0038] Figure 8 The figure is a chart of the immunohistochemical EBER detection results of the nasopharyngeal carcinoma tissue and the cultured nasopharyngeal carcinoma organoid in Example 1. DETAILED DESCRIPTION

[0039] The application will be further described in detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0040] The chemical reagents used in the following examples are shown in Table 1 below: Table 1

[0041] Preparation and preservation of ex vivo active nasopharyngeal carcinoma tissue: Obtain the approval of the Ethics Committee of the Second People's Hospital of Shenzhen (the First Affiliated Hospital of Shenzhen University) and the Second People's Hospital of Shenzhen, and for the clinical population with nasopharyngeal carcinoma, after following their consent, take the nasopharyngeal carcinoma living tissue under the nasal endoscope, obtain the ex vivo nasopharyngeal carcinoma tissue, and place it in the tissue sample preservation solution (50% Advanced DMEM / F-12, 40% FBS, 10% DMSO) for low-temperature preservation, or send it to the laboratory for standby.

[0042] The recombinant LMP2 expression protein used is prepared by the following method: The LMP2 gene fragment (the sequence is shown in SEQ ID NO. 3) is artificially synthesized by a gene company, and then the LMP2 gene fragment and the pET28a vector are double-digested with BamH I and Hind III two restriction endonucleases (the enzyme digestion reaction conditions are as follows: LMP2 gene fragment / pET28a vector, 10 μL; restriction endonuclease BamHI, 1 μL; restriction endonuclease Hind III, 1 μL; 10×buffer, 5 μL; ddH2O, 15 μL). After enzyme digestion, 1% nucleic acid gel electrophoresis purification is performed, the purified target fragment is connected with the empty vector, and then transformed into competent cells DH5α, which is identified as positive by PCR, and sent to the gene company for sequencing to be correct, to obtain the recombinant plasmid. Then the recombinant plasmid is transformed into BL21 (DE3) competent cells for expression (conditions are as follows: take 1 ml of recombinant plasmid and mix with 100 μL of BL21 (DE3) competent cells in ice bath for 30 min, 42°C water bath for 90 s, and ice bath for 2 min cooling; then add 1 ml of LB liquid medium, 37°C shaking bed 200 r / min vibration culture 1 h; 100 μL of bacterial liquid is absorbed and coated on ampicillin-resistant LB plate, 37°C overnight culture; pick single colony on LB plate medium, inoculate in 100 mg / ml ampicillin-resistant LB liquid medium for amplification culture, and when the bacterial liquid culture reaches OD600 value of 12, add IPTG with a final concentration of 0.1 mM, 37°C induction for 4 h, collect the fermentation broth and centrifuge for 30 min, collect the bacterial precipitate, resuspend the bacterial precipitate with buffer B, and then lyse by high-pressure homogenizer, collect the supernatant by centrifugation, and purify by nickel ion affinity chromatography), to obtain the recombinant LMP2 expression protein. Example

[0043] 1. Preparation of active nasopharyngeal carcinoma cells: The ex vivo nasopharyngeal carcinoma tissue is taken out from its sample preservation solution, pre-cooled, then washed with PBS, cut into small pieces, and digested with tissue digestion solution (5 ml Advanced DMEM / F-12 + 50 mg / ml collagenase type II + 20 mg / ml deoxyribonuclease I + 100 mg / ml TrypLE Express + 50 μg / ml Y27632) at 37°C for 1 h, then centrifuged, and the supernatant is discarded to obtain the first precipitate. Add 3 ml of 0.25% trypsin and digest at 37°C for 10 min. Stop the digestion with 20% FBS Advanced DMEM / F-12 medium, centrifuge, discard the supernatant, and obtain the second precipitate; thus the active nasopharyngeal carcinoma cells are obtained.

[0044] 2. Preparation of culture medium for nasopharyngeal carcinoma organoids, with the following components added: Antibiotic-Antimycotic, 60 μmol / mL; Gluta MAX, 20 nmol / L; B27 supplement, 1.0% by volume; N-acetylcysteine, 0.25 mg / ml; Nicotinamide, 10 nmol / L; p38 MAPK inhibitor (SB202190), 10 μmol / mL; TGF-ß type I receptor inhibitor (A83-01), 10 nmol / L; Recombinant human R-type response protein (R-Spondin1), 5000 ng / mL, Recombinant human noggin protein, 100 ng / mL, Recombinant LMP2 expression protein, 200 ng / mL Compound Y27632, 20 μmol / mL; FGF-10, 50 μmol / mL; EGF, 50 nmol / mL; 3. Culture steps of nasopharyngeal carcinoma organoids: (1) Disperse the nasopharyngeal carcinoma tissue to obtain active nasopharyngeal carcinoma cells; specifically including: resuspend the nasopharyngeal carcinoma cells of step 1 with 20% FBS Advanced DMEM / F-12, filter through a 100 μm molecular sieve, centrifuge the filtrate, and discard the supernatant to obtain the single-cell third precipitate.

[0045] (2) resuspend the resuspended nasopharyngeal carcinoma cells with Advanced DMEM / F-12 containing multiple trace elements and Matrigel matrix glue in a mixture with a volume ratio of 5:1; to obtain target nasopharyngeal carcinoma cells; specifically comprising: resuspending the third precipitate with Advanced DMEM / F-12 medium and Matrigel matrix glue (volume ratio of 5:1), inoculating, and incubating and culturing.

[0046] (3) inoculate the target nasopharyngeal carcinoma cells into the medium for nasopharyngeal carcinoma organoids for culture, and replace the components of the medium to the initial state every 3-5 days to achieve the culture of nasopharyngeal carcinoma organoids.

[0047] The above digestion and inoculation process of nasopharyngeal carcinoma tissue is shown in Figure 1 . Figure 1 In the figure, A is the cut nasopharyngeal carcinoma tissue, B is the image of the cut carcinoma tissue before digestion after adding tissue digestion solution, C is the image of the first precipitate after digestion with tissue digestion solution and addition of trypsin, and D is the image of the gel drop shape inoculated in the six-well plate.

[0048] The above culture process of nasopharyngeal carcinoma organoids is shown in Figure 2 : wherein, A is the organoid cultured for 0 days; B is the organoid cultured for 5 days; C is the organoid cultured for 10 days; D is the organoid cultured for 14 days.

[0049] The nasopharyngeal carcinoma organoids obtained in this example were identified: the nasopharyngeal carcinoma organoids obtained by culture were subjected to hematoxylin-eosin staining together with the prepared nasopharyngeal carcinoma tissue sections, to obtain the results shown in Figure 3 . Figure 3 In the figure, A is the HE staining picture of the nasopharyngeal carcinoma tissue of this example, and B is the HE staining picture of the nasopharyngeal carcinoma organoids cultured in this example. From the Figure 3 results, it can be seen that the obtained nasopharyngeal carcinoma organoids have a certain three-dimensional morphology, indicating that the organoids have been successfully obtained.

[0050] The nasopharyngeal carcinoma organoids obtained in this example were subjected to immunohistochemical staining test, to obtain the results shown in Figure 4The results are shown in the figure; wherein, A is the picture of LMP1 staining of nasopharyngeal carcinoma organoids, B is the picture of AE1 / AE3 staining of nasopharyngeal carcinoma organoids, and C is the picture of Vimentin staining of nasopharyngeal carcinoma organoids. The identification markers of immunohistochemical staining include: AE1 / AE3; LMP1; Vimentin. The specific staining steps refer to the standard of tissue section immunohistochemistry and tissue section immunofluorescence experiment. The organoids have specific proteins of the cells inside the nasal cavity, which indicates that the nasopharyngeal carcinoma organoids are successfully cultured. The nasopharyngeal carcinoma tissue and the cultured nasopharyngeal carcinoma organoids in this example are subjected to immunohistochemistry and EBER test, and the results are shown in the figure. Figures 5-8 It can be seen from the above results that the nasopharyngeal carcinoma organoids obtained by the present application can better maintain the specificity of the tissue cells.

[0051] 4. A subculture method of nasopharyngeal carcinoma organoids (1) Collect the nasopharyngeal carcinoma organoids with good growth state after being cultured for 2 weeks together with Matrigel, centrifuge at 300 g for 5 min at room temperature, discard the supernatant culture solution, and obtain the first precipitate containing Matrigel.

[0052] (2) Add 3 ml of 0.25% trypsin, and digest at 37°C for 8 min to obtain nasopharyngeal carcinoma cells, and continue to subculture using the above-mentioned culture medium for nasopharyngeal carcinoma organoids. Example

[0053] The difference between this example and Example 1 is that the content of FGF-10 in the culture medium for nasopharyngeal carcinoma organoids is adjusted to 30 μmol / mL, and the content of EGF is adjusted to 30 nmol / mL. Example

[0054] The difference between this example and Example 1 is that the content of recombinant human R-type response protein (R-Spondin1) in the culture medium for nasopharyngeal carcinoma organoids is adjusted to 2000 ng / mL, the content of recombinant human noggin (Noggin recombinant protein) is adjusted to 70 ng / mL, and the content of recombinant LMP2 expression protein is adjusted to 100 ng / mL.

[0055] Comparative Example 1 The difference between this comparative example and Example 1 is that FGF-10 is not added to the culture medium for nasopharyngeal carcinoma organoids.

[0056] Comparative Example 2 The difference between this comparative example and Example 1 is that EGF is not added to the culture medium for nasopharyngeal carcinoma organoids.

[0057] Comparative Example 3 The present comparative example differs from Example 1 in that the compound Y27632 is not added to the medium for the nasopharyngeal carcinoma organoids.

[0058] Comparative Example 4 The present comparative example differs from Example 1 in that the recombinant human R-spondin 1 is not added to the medium for the nasopharyngeal carcinoma organoids.

[0059] Comparative Example 5 The present comparative example differs from Example 1 in that the recombinant human noggin is not added to the medium for the nasopharyngeal carcinoma organoids.

[0060] Comparative Example 6 The present comparative example differs from Example 1 in that the recombinant LMP2 expression protein is not added to the medium for the nasopharyngeal carcinoma organoids.

[0061] The culture period (the period for which the medium of the examples and comparative examples was used for the culture of the nasopharyngeal carcinoma organoids was recorded from the time of inoculation into the medium), the passage number (the medium of the examples and comparative examples was used for the passage culture of the nasopharyngeal carcinoma organoids obtained in Example 1, the shape of the nasopharyngeal carcinoma organoids was detected, and the final passage number was obtained until the organoids could not be successfully passaged), and the passage success rate (20 different nasopharyngeal carcinoma tissue samples were used for the organoid culture and passage culture on the medium of the examples and comparative examples, and the percentage of the first passage (P1) success was recorded) of the medium obtained in the above examples and comparative examples were detected, and the results are shown in Table 2 below.

[0062] Table 2

[0063] From the results in Table 2, the following conclusions can be drawn: through the combined action of the recombinant human R-spondin, the recombinant human noggin, the recombinant LMP2 expression protein, and the ROCK inhibitor Y27632, FGF-10, and hEGF, the present application can achieve the effects of shortening the culture period, increasing the passage number, and improving the passage success rate. The absence of any one of the components will result in a significant decrease in the culture effect of the nasopharyngeal carcinoma organoids.

[0064] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A medium for culturing nasopharyngeal carcinoma organoids, characterized in that, The functional components include the following content ranges: antibiotics, 30-120 μmol / mL; glutamine supplement, 15-25 nmol / L; B27 serum-free additive, 0.5%-1.5% by volume; N-acetylcysteine, 0.2-0.3 mg / ml; nicotinamide, 8-12 nmol / L; p38 MAPK inhibitor, 7-12 μmol / mL; TGF-β type I receptor inhibitor, 7-12 nmol / L; recombinant human R-type response protein, 400-6000 ng / mL, recombinant human noggin, 70-130 ng / mL, recombinant LMP2 expression protein, 100-500 ng / mL, ROCK inhibitor, 10-30 μmol / mL; fibroblast growth factor 10 (FGF-10), 30-60 μmol / mL; epidermal growth factor (EGF), 30-60 nmo l / mL.

2. The medium for culturing nasopharyngeal carcinoma organoids according to claim 1, characterized in that, The antibiotics include one or more of penicillin, streptomycin, and amphotericin B; the p38 MAPK inhibitor includes one or more of 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole, compound SB203580, compound SB20219, and compound SB239063; and the TGF-β type I receptor inhibitor includes one or more of compound A83-01, compound SB431542, and compound LY2157299.

3. The medium for culturing nasopharyngeal carcinoma organoids according to claim 1, characterized in that, The amino acid sequence of the recombinant human R-type response protein is shown in SEQ ID NO. 1; and the amino acid sequence of the recombinant human noggin is shown in SEQ ID NO.

2.

4. The medium for culturing nasopharyngeal carcinoma organoids according to claim 1, characterized in that, The recombinant LMP2 expression protein is prepared as follows: The LMP2 gene is cloned into a pET28a vector to obtain a recombinant plasmid, which is then transformed into BL21 competent cells for expression to obtain the recombinant LMP2 expression protein.

5. The medium for culturing nasopharyngeal carcinoma organoids according to claim 1, characterized in that, The ROCK inhibitor includes one or more of compound Y-27632, compound fasudil, hydroxyfasudil, compound Y-33075, compound T14960, GSK429286A, and N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide.

6. The medium for culturing nasopharyngeal carcinoma organoids according to claim 1, characterized in that, The ROCK inhibitor is compound Y-27632; the molar ratio of FGF-10, EGF and compound Y27632 is 1 : (5 x 10 -4 -2 x 10 -3 ): (0.2-1.0).

7. A method for culturing a nasopharyngeal carcinoma organoid, characterized by, The method includes the following steps: (1) dispersing an ex vivo active nasopharyngeal carcinoma tissue in a tissue digestion solution and a trypsin solution in sequence for treatment to obtain an active nasopharyngeal carcinoma cell; (2) resuspending the nasopharyngeal carcinoma cell of step (1) in a serum-reduced basal medium, filtering with a molecular sieve, and centrifuging to obtain a resuspended nasopharyngeal carcinoma cell from the precipitate; (3) resuspending the resuspended nasopharyngeal carcinoma cell of step (2) in a mixture of a serum-reduced basal medium and Matrigel to obtain a target nasopharyngeal carcinoma cell, which is then inoculated into the medium for nasopharyngeal carcinoma organoids of any one of claims 1-6 for culture, and the components of the medium are updated to the initial state at intervals, and a nasopharyngeal carcinoma organoid is obtained by culture.

8. The method of culturing a nasopharyngeal carcinoma organoid according to claim 7, wherein, The tissue digestion solution in step (1) comprises 40-60 mg / ml collagenase type II, 18-22 mg / ml deoxyribonuclease I, 95-105 mg / ml TrypLE Express and 45-55 μg / ml Y-27632; and the trypsin solution has a mass concentration of 0.1%-0.25%.

9. The method of culturing a nasopharyngeal carcinoma organoid according to claim 7, wherein, In the mixture in step (3), the volume ratio of serum-reduced basal medium to Matrigel is 3-10:1; and in step (5), the component of the interval update medium is updated to the initial state at an interval of 3-5 days.

10. A nasopharyngeal carcinoma organoid, which is cultured by the method of any one of claims 7-9.

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