Oral squamous cell carcinoma neck lymph node metastasis organoid culture medium and construction method

Abstract

The application relates to the field of organoids, and discloses a culture medium and a construction method of an oral squamous carcinoma neck lymph node metastasis organoid. The method comprises the following steps: digesting tumor tissue in a digestive solution to obtain a digestive tissue suspension, performing cell filtration centrifugation on the digestive tissue suspension, retaining a cell precipitate, and obtaining the cell precipitate; resuspending the cell precipitate by using a buffer solution, and counting live cell numbers; inoculating organoids into a well plate according to the counted live cell numbers, adding a culture medium, and placing the well plate in a culture box for culture; after 3 days of culture, only 5% active cells of tumor infiltrating lymphocytes in the organoid culture system are left, the organoids are digested, the digestion is terminated, and centrifugation is performed. The application solves the problem that lymphocytes have an inhibitory effect on organoid formation in organoid culture, and affect the success rate of tumor organoid construction.

Description

TECHNICAL FIELD

[0001] The application relates to the field of organoids, and in particular to a culture medium and a construction method of an oral squamous carcinoma neck lymph node metastasis organoid. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC) is a common malignant tumor in the oral and maxillofacial region. Large-scale sequencing data has revealed that genetic variations in oral squamous carcinoma are concentrated in tumor suppressor genes, and the mutation frequency of proto-oncogenes is extremely low, and there are few effective therapeutic targets. There is still direct and effective evidence between sequencing data guiding clinical drug use and actual drug efficacy of patients, and new treatment methods are urgently needed to provide individualized treatment plans for patients.

[0003] An organoid is a kind of in vitro cell model that 3D cultures cells with stem cell potential. The organoid can spontaneously simulate the microstructure and function of the in vivo organ, and the genomic characteristics of the organoid are similar to the in vivo primary tumor with a similarity of about 90%. Moreover, the organoid can maintain the heterogeneity of tumor cells in the patient's primary tumor. The tumor organoid model is a widely used drug sensitivity screening and verification model in current preclinical research. However, due to the bacterial contamination in the oral cavity, this is one of the main factors that reduces the success rate of oral cancer organoid construction.

[0004] Oral cancer neck lymph node metastasis is one of the most common symptoms of oral cancer, and neck lymph node metastasis is also one of the main risk factors for oral cancer prognosis. Oral cancer neck lymph node metastasis is more invasive and has stronger stemness than oral cancer primary tumor cells, and is more suitable for organoid culture. For a considerable portion of early oral cancer patients (T1 and T2), the initial surgery often adopts simple primary tumor resection, but about 30% of these patients often develop neck lymph node metastasis after surgery. For these patients with metastasis, constructing their lymph node metastasis organoids can provide postoperative adjuvant drug basis for subsequent drug sensitivity detection, and there is a very broad application scenario. At the same time, it is still necessary to mention that about 5% of head and neck lymph node metastasis patients have unknown primary tumors, and the subsequent treatment of these patients can only rely on the relevant information of the metastatic lymph nodes to develop subsequent treatment plans. The neck lymph node is in a sterile environment, avoiding the adverse factors of contamination in the construction process of the primary tumor organoid of oral cancer. Therefore, constructing the organoid of the lymph node metastasis of oral cancer provides a basis for individualized drug use for patients, and has a very broad application demand. However, the oral squamous carcinoma neck lymph node contains a large number of lymphocytes, which inhibit the formation of organoids in organoid culture, affecting the success rate of tumor organoid construction. SUMMARY

[0005] The main objective of this invention is to solve the problem that existing technologies cannot effectively construct organoid models of lymph node metastases from oral squamous cell carcinoma due to the presence of lymphocytes.

[0006] The first aspect of this invention provides a culture medium for constructing an organoid model of oral squamous cell carcinoma lymph node metastases. The culture medium is prepared in Advanced DMEM / F12 basal medium with the following substances added in weight percentage: 1-2% Hepes buffer, 1-2% penicillin-streptomycin solution, 1-2% Glutamax, 2-3% B27, 1-2% N2, 5-10 mmol / L nicotinamide, 10-20 ng / mL NRG1, 1-2 mmol / L N-acetylcysteine, 10-20 μM Y-27632, 100-120 ng / mL Noggin, 100-120 ng / mL R-spondin1, 500-550 nmol / L A83-01, 100-120 ng / mL EGF, 50-60 ng / mL FGF10, 100-120 ng / mL FGF2, and 10-15 μM Forskolin.

[0007] This invention also provides a method for constructing an organoid model of oral squamous cell carcinoma lymph node metastases, the method comprising the following steps:

[0008] The tumor tissue was digested in digestive fluids to obtain a suspension of digested tissue.

[0009] The digested tissue suspension was subjected to cell filtration and centrifugation, and the precipitate was retained to obtain cell precipitate;

[0010] The cell pellet was resuspended in buffer solution, and the number of viable cells was counted.

[0011] Based on the count of live cells, organoids were seeded into well plates, culture medium was added, and the well plates were placed in an incubator for incubation.

[0012] After 3 days of culture, only 5% of the tumor-infiltrating lymphocytes remained viable in the organoid culture system. After digesting the organoids, digestion was stopped and the cells were centrifuged.

[0013] The cell pellet was resuspended in buffer solution, and the number of viable cells was counted.

[0014] Based on the count of live cells, organoids were seeded into well plates, culture medium was added, and the well plates were placed in an incubator for incubation.

[0015] The culture medium is added with the following substances in weight percentage under the Advanced DMEM / F12 basic medium: 1-2% of Hepes buffer, 1-2% of penicillin streptomycin solution, 1-2% of Glutamax, 2-3% of B27, 1-2% of N2, 5-10 mmol / L of nicotinamide, 10-20 ng / mL of NRG1, 1-2 mmol / L of N-acetyl cysteine, 10-20 uM of Y-27632, 100-120 ng / ml of Noggin, 100-120 ng / ml of R-spondin1, 500-550 nmol / L of A83-01, 100-120 ng / ml of EGF, 50-60 ng / ml of FGF10, 100-120 ng / ml of FGF2, 10-15 uM of Forskolin.

[0016] The application selects a culture medium with specific properties, and when the tumor infiltrating lymphocytes only have 5% of active cells left, the organoids are digested again, so that the success rate can be increased to 75%. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The photo is of the primary tumor single cell culture on the first day of Example 1;

[0018] Figure 2 The photo is of the primary tumor single cell culture after 3 days of Example 1, which is subcultured and continues to be cultured for 4 days;

[0019] Figure 3 The vitality comparison before and after the immune cell culture for 3 days

[0020] Figure 4 The photo is of the primary tumor single cell culture on the first day of Example 2;

[0021] Figure 5 The photo is of the primary tumor single cell culture for 7 days of Example 2.

[0022] Figure 6 The graph is of the organoid diameter comparison of Example 1 and Example 2, left for Example 1 and right for Example 2. DETAILED DESCRIPTION

[0023] The terms "first", "second", "third", "fourth" and the like in the description and in the claims of the present application, and above-mentioned drawings, if any, are used to distinguish between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the use of the terms so-termed "first", "second", "third", "fourth" and the like, if any, in the description and in the claims of the present application is not used to designate a certain order or chronology, but to distinguish between similar objects. It is to be understood that the data so used can be interchanged, where appropriate, so that the embodiments described herein can be carried out in an order other than the one illustrated or described herein. Furthermore, the terms "comprising" or "having" and any variations thereof are intended to cover the non-exclusive inclusion of the steps or elements described, for example, of a process, method, system, product or apparatus, and that the process, method, system, product or apparatus can include additional steps or elements not expressly listed or inherent to such process, method, system, product or apparatus.

[0024] Raw material sources:

[0025] Advanced DMEM / F12 medium purchased from (Gibco) Hepes buffer purchased from (Gibco) Penicillin streptomycin solution purchased from (Gibco) 1% Glutamax purchased from (Sigma) 2% B27 purchased from (Gibco) 1% N2, 5 mmol / L nicotinamide purchased from (MCE) 10 ng / mL NRG1 purchased from (MCE) 1 mmol / L N-acetyl cysteine purchased from (Sigma) 10 uM Y-27632 purchased from (MCE) 100 ng / ml Noggin purchased from (MCE) 100 ng / ml R-spondin1 purchased from (MCE) 500 nmol / L A83-01 purchased from (MCE) 100 ng / ml EGF purchased from (MCE) 50 ng / ml FGF10 purchased from (MCE) 100 ng / ml FGF2 purchased from (MCE) 10 uM Forskolin purchased from (MCE).

[0026] Example 1

[0027] Oral squamous cell carcinoma lymph node metastasis organoid culture method:

[0028] S1, Digestive enzyme configuration: 5g / ml collagenase I and 10ug / ml DNAase I with serum-free DMEM medium as solvent, stored at 4 degrees Celsius;

[0029] Washing solution configuration: 4% BSA with PBS as solvent, 0.22um filter core filtration sterilization, stored at 4 degrees Celsius;

[0030] Matrigel matrix was melted on ice;

[0031] S2, configure the OSCC lymph node metastasis organoid culture medium, the components are as follows:

[0032] The basal culture medium consisted of Advanced DMEM / F12 medium, 1% Hepes buffer, 1% penicillin-streptomycin solution, 1% Glutamax, 2% B27, 1% N2, 5 mmol / L nicotinamide, 10 ng / mL NRG1, 1 mmol / L N-acetylcysteine, 10 μM Y-27632, 100 ng / mL Noggin, 100 ng / mL R-spondin1, 500 nmol / L A83-01, 100 ng / mL EGF, 50 ng / mL FGF10, 100 ng / mL FGF2, and 10 μM Forskolin. The concentrations of each component of the specific factors were based on their concentrations in OSCC organoid culture medium.

[0033] S3. Obtaining samples of oral squamous cell carcinoma cervical lymph node metastases through clinical operation: Obtain samples of oral squamous cell carcinoma cervical lymph node metastases through surgical resection. Take 0.5cm × 0.5cm × 0.5cm tissue, rinse 3 times with sterile physiological saline, put the tissue into a 15ml centrifuge tube, and store and transport it on ice in pre-cooled DMEM medium containing 20% ​​penicillin and antibiotics.

[0034] S4. Tissue pretreatment: Rinse the tumor tissue from step S3 three times with sterile PBS buffer, place the tissue in a 5ml centrifuge tube, and cut the tissue into multiple small tissue blocks of 3mm × 3mm × 3mm using sterile ophthalmic scissors.

[0035] S5. Tissue block digestion: Add 5 ml of the digestion solution prepared in step S1 to the centrifuge tube containing the shredded tissue block from step S4. Digest at 37°C and 400 rpm in a constant temperature shaker for 15 minutes. Filter the mixture through a 40 μm filter to obtain a single-cell suspension. Centrifuge at 1500 rpm for 5 minutes at 4°C. After centrifugation, remove the supernatant and add 5 ml of the washing solution prepared in step S1. Repeat centrifugation and remove the supernatant.

[0036] S6. Add the cell clusters obtained in step S5 to 2 ml of erythrocyte lysis buffer, and gently pipette the precipitated cell clusters with a 1 ml pipette tip to resuspend them for 2 minutes. Transfer the cell suspension to a 15 ml centrifuge tube, add 10 ml of washing buffer, and centrifuge at 1500 rpm for 5 minutes at 4 degrees Celsius. Discard the supernatant after centrifugation.

[0037] S7. Add the cell clusters obtained in step S6 to 1 ml of washing solution, gently pipette the precipitated cell clusters with a 1 ml pipette tip to resuspend them, and count the cells.

[0038] S8, centrifuge the cell suspension obtained in step S7 at 1500 rpm for 5 minutes at 4 degrees, and remove the supernatant after centrifugation. According to the total number of cells calculated in step S7, add the corresponding volume of melted Matrigel to make the final volume of cells 5000 / ul, gently blow the precipitated cell mass with a 100ul gun head to resuspend it, gently blow the precipitated cell mass with a truncated gun head to resuspend it, use a 2.5ul gun head to suck 2ul of cell suspension into a 96-well plate, and place it in a cell incubator for 10 minutes to allow the Matrigel to fully solidify. After the Matrigel solidifies, add 200ul of the medium in step S2, and place it back in the incubator for further culture. Observe the tumor organoids on the second day, and some tumors should have formed spherical structures. Replace the fresh medium every 3 days;

[0039] S9, after 3 days of culture, the tumor infiltrating lymphocytes in the organoids cultured in the 96-well plate in step S8 only have 5% active cells left, digest the organoids with Trapline, terminate the digestion with DMEM after 3 minutes, centrifuge, count, and according to the total number of cells, add the corresponding volume of melted Matrigel to make the final volume of tumor cells 50000 / ul, gently blow the precipitated cell mass with a 100ul gun head to resuspend it, gently blow the precipitated cell mass with a truncated gun head to resuspend it, use a 2.5ul gun head to suck 2ul of cell suspension into a 96-well plate, and place it in a cell incubator for 10 minutes to allow the Matrigel to fully solidify. After the Matrigel solidifies, add 200ul of the medium in step S2, and place it back in the incubator for further culture. Observe the tumor organoids on the second day, and some tumors should have formed spherical structures. Replace the fresh medium every 3 days. After 4 days of continuous culture, a large number of tumor organoids can be seen in the Matrigel.

[0040] The experimental results are shown in Figure 1 、 Figure 2 and Figure 3

[0041] Example 2

[0042] Oral squamous carcinoma neck lymph node metastasis organoid culture method:

[0043] S1, prepare digestive enzymes: 5g / ml collagenase I and 10ug / ml DNAase I in serum-free DMEM medium as solvent, store at 4 degrees Celsius;

[0044] Prepare the washing solution: 4% BSA in PBS as solvent, filter sterilization with a 0.22um filter, store at 4 degrees Celsius;

[0045] Melt Matrigel on ice;

[0046] S2, the medium of embodiment 1 is configured;

[0047] S3, clinical operation obtains oral squamous cell carcinoma neck lymph node metastasis sample: oral squamous cell carcinoma neck lymph node metastasis sample is obtained by surgical resection, 0.5cm x 0.5cm x 0.5cm tissue is cut, and the tissue is washed with sterile normal saline for 3 times, and the tissue is placed in a 15ml centrifuge tube containing 20% double-antibody pre-cooled DMEM medium, and is stored and transported on ice;

[0048] S4, tissue pretreatment: the tumor tissue of step S3 is washed with sterile PBS buffer for 3 times, the tissue is placed in a 5ml centrifuge tube, and the tissue is cut into 3mm x 3mm x 3mm small tissue blocks by using sterile ophthalmic scissors;

[0049] S5, tissue block digestion: 5ml of the digestion solution configured in step S1 is added to the centrifuge tube of the cut tissue block in step S4, and the digestion is carried out at 37℃ temperature and at a rotation speed of 400rpm in a constant temperature shaker for 15 minutes; the single cell suspension is obtained by filtering with a 40um filter screen, and is centrifuged at 4 degree temperature and at a rotation speed of 1500rpm for 5 minutes; after centrifugation, the supernatant is removed, and 5ml of the washing solution configured in step S1 is added, and the centrifugation is repeated to remove the supernatant.

[0050] S6, the cell mass obtained in step S5 is added into 2ml of red blood cell lysis solution, 1ml of gun head is gently blown to resuspend the precipitated cell mass, and the time is counted for 2 minutes. The cell suspension is transferred to a 15ml centrifuge tube, 10ml of washing solution is added, and centrifugation is carried out at 4 degree temperature and at a rotation speed of 1500rpm for 5 minutes, and after centrifugation, the supernatant is removed.

[0051] S7, the cell mass obtained in step S6 is added into 1ml of washing solution, 1ml of gun head is gently blown to resuspend the precipitated cell mass, and the cell count is calculated.

[0052] S8, the cell suspension obtained in step S7 is centrifuged at 4 degree temperature and at a rotation speed of 1500rpm for 5 minutes, and after centrifugation, the supernatant is removed. According to the total number of cells calculated in step S7, a corresponding volume of melted matrix glue Matrigel is added, so that the final volume of cells is 5000 / ul, 100ul of gun head is gently blown to resuspend the precipitated cell mass, the precipitated cell mass is gently blown to resuspend by using a truncated gun head, 2ul of cell suspension is taken by using 2.5ul of gun head and is dropped into a 96-well plate, and is placed into a cell culture box for 10 minutes to allow the matrix glue (Matrigel) to be fully solidified. After the matrix glue (Matrigel) is solidified, 200ul of the medium in step S2 is added, and is placed back into the culture box for continuous culture. Fresh medium is replaced every 3 days; after 5-7 days of tumor organ culture, a small amount of tumor organ can be observed.

[0053] The experimental results are shown in Figure 4 andFigure 5 .

[0054] Effect of experimental examples on the success rate of OSCC culture

[0055] In this experimental example, 8 OSCC organoid samples were included in each group (Examples 1 and 2). According to the medium formula of Example 1, the OSCC organoids obtained after the culture operation steps of Examples 1 and 2 were detected. The success rates of the two groups of cultured OSCC organoids are shown in Table 1. The success of culture refers to the fact that the tumor cells or cell clusters after digestion can form a three-dimensional structure in vitro and the size of the organoid continues to grow, which can be identified as successful organoid culture.

[0056] Table 1

[0057]

[0058] According to the above results, the OSCC organoids obtained after the culture operation steps of Example 1 can be successful in 6 cases, and the success rate can be increased to 75% (6 / 8). The effect of Example 2 organoid culture is not as good as that of Example 1, only reaching 12.5% (1 / 8).

[0059] The above-described and the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for constructing an oral squamous carcinoma cervical lymph node metastasis organoid, the method comprising the following steps: digesting an oral squamous carcinoma cervical lymph node metastasis sample tissue in a digestive solution to obtain a digestive tissue suspension, carrying out cell filtration centrifugation on the digestive tissue suspension to retain a cell precipitate, and obtaining the cell precipitate; resuspending the cell precipitate with a buffer solution and counting the number of live cells; according to the counted number of live cells, inoculating an organoid into a well plate, adding a culture medium, and placing the well plate in an incubator for culture; after 3 days of culture, only 5% of active cells of tumor infiltrating lymphocytes in the organoid culture system remain, and the organoid is digested, the digestion is terminated, and centrifugation is carried out; resuspending the cell precipitate with a buffer solution and counting the number of live cells; according to the counted number of live cells, inoculating an organoid into a well plate, adding a culture medium, and placing the well plate in an incubator for culture to obtain an oral squamous carcinoma cervical lymph node metastasis organoid; the culture medium is added with the following components on the basis of Advanced DMEM / F12 basic culture medium: 1-2% Hepes buffer solution, 1-2% penicillin streptomycin solution, 1-2% Glutamax, 2-3% B27, 1-2% N2, 5-10 mmol / L nicotinamide, 10-20 ng / mL NRG1, 1-2 mmol / L N-acetyl cysteine, 10-20 uM Y-27632, 100-120 ng / ml Noggin, 100-120 ng / ml R-spondin1, 500-550 nmol / L A83-01, 100-120 ng / ml EGF, 50-60 ng / ml FGF10, 100-120 ng / ml FGF2, and 10-15 uM Forskolin.

2. The method according to claim 1, wherein, the digestive solution is composed of 5 g / ml collagenase I and 10 ug / ml DNase I, and the digestive solution is dissolved in a serum-free DMEM culture medium.

3. The method for constructing an organoid from cervical lymph node metastases of oral squamous cell carcinoma according to claim 1, characterized in that, the buffer solution is a sterile PBS buffer solution.

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