CN117821386A - Culture medium for nasopharyngeal carcinoma organoid culture, and culture method and application thereof - Google Patents
Culture medium for nasopharyngeal carcinoma organoid culture, and culture method and application thereof Download PDFInfo
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- CN117821386A CN117821386A CN202211190924.3A CN202211190924A CN117821386A CN 117821386 A CN117821386 A CN 117821386A CN 202211190924 A CN202211190924 A CN 202211190924A CN 117821386 A CN117821386 A CN 117821386A
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Abstract
The invention provides a culture medium for nasopharyngeal carcinoma organoids, a culture method and application thereof. The nasopharyngeal carcinoma organoid medium of the present invention comprises an epidermal growth factor, a hepatocyte growth factor, an ITS cell culture additive, insulin, a Rho protease inhibitor, dexamethasone, neuregulin 1, sodium pyruvate, fetal bovine serum, a fibroblast growth factor, an insulin-like growth factor, and an additive selected from at least one of a B27 additive and an N2 additive. The nasopharyngeal carcinoma organoid culture medium can realize the effective and rapid amplification of the nasopharyngeal carcinoma organoid, so that the organoid obtained by amplification maintains the pathological characteristics of a patient, the culture success rate and the amplification rate of the nasopharyngeal carcinoma organoid are improved, and a research foundation can be provided for the personalized treatment of the patient.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium and a culture method for nasopharyngeal carcinoma organoids and application thereof in evaluation and screening of curative effects of medicines.
Background
Nasopharyngeal carcinoma is a generic term for malignant tumors that occur in the nasopharynx. Nasopharyngeal carcinoma includes cancers that are primarily in the nasopharyngeal tongue (2/3 of the anterior lingual), buccal mucosa, gums, bottom of the mouth, hard palate, one of the more common malignant tumors in the benign region (head and neck) of the sixth largest malignancy worldwide, the second most common malignancy in the head and neck. Nasopharyngeal carcinoma can occur at any age, and the incidence peak is 40-60 years old, and the composition ratio of men and women is about 2:1. In recent years, nasopharyngeal carcinoma presents the following new characteristics: firstly, the incidence rate of tongue cancer increases more rapidly, which is nearly half of all nasopharyngeal carcinoma; secondly, the disease age of nasopharyngeal carcinoma has a tendency of younger age, and the young patients of 20-30 years old are not rare; third, female patients are increasing year by year. Although the traditional treatment methods including operation, radiotherapy and chemotherapy have different degrees of development in the past 20 years, the prognosis of nasopharyngeal carcinoma is still unsatisfactory, the survival rate of about 50-60% in 5 years is only about 1/3 of patients can relapse, and swallowing, language function, facial appearance and the like of the patients subjected to the operation treatment are seriously affected, so that obvious and serious personal injuries, eating and language barriers exist. The main reasons for affecting survival are local recurrence and lymphatic metastasis. Thus, new therapeutic approaches such as targeted tumor therapy and other biological therapies are receiving increasing attention. Meanwhile, how to improve the anticancer efficacy of the drug treatment and reduce the toxic and side effects of the drug, namely the targeting of the anticancer treatment, is a concern of the basic and clinical research of antitumor.
The traditional clinical drug sensitivity detection mostly adopts two-dimensional cell culture. However, two-dimensional cultured cells simulate tissue physiological conditions only to a limited extent, lack of a real tissue structure in vivo, are prone to low differentiation level and loss of cell physiological functions, and further lead to the difficulty in predicting clinical actual results from the obtained experimental results. Organoids, which belong to three-dimensional (3D) cell cultures, are mainly derived from embryonic stem cells, induced pluripotent stem cells and adult stem cells, which have the ability to differentiate in humans. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of each organ. Under certain induction conditions in vitro, these stem cells can self-organize into a mini-structure with a diameter of only a few millimeters. Tumor organoids were obtained from primary tumors in patients and some miniature 3D tumor cell models were developed in the laboratory. The tumor organoids highly simulate the characteristics of the source tumor tissues, retain tumor heterogeneity among individuals, and can be used for functional tests, such as high-throughput drug screening and personalized accurate treatment.
At present, the nasopharyngeal carcinoma organoid culture method mostly adopts R-spondin-1, noggin and some expensive protein factors, so that the organoid culture cost is high; and the current organoid culture method has complex operation and large technical difficulty, so that the large-scale commercial application of the organoid culture method is limited. Therefore, there is a need to develop a low cost, simple and highly successful organoid culture method and medium.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture medium and a culture method for rapidly amplifying nasopharyngeal carcinoma organoids in vitro.
In one aspect, the invention provides a nasopharyngeal carcinoma organoid medium comprising Epidermal Growth Factor (EGF), hepatocyte Growth Factor (HGF), ITS cell culture additive, insulin, rho protease inhibitor, dexamethasone, neuregulin 1 (NRG 1), sodium pyruvate, fetal Bovine Serum (FBS), fibroblast growth factor (bFGF), insulin-like growth factor number one (IGF-1), and at least one cell culture additive selected from the group consisting of N2 and B27.
In an embodiment of the invention, the content of each component in the culture medium of the invention satisfies any one or more or all of the following:
(1) The concentration range of the EGF is preferably 0.6-45 ng/mL;
(2) The concentration range of the hepatocyte growth factor HGF is preferably 1-100 ng/mL;
(3) The volume ratio of ITS cell culture additive to the culture medium is preferably 1:25-1:400;
(4) The concentration range of insulin is preferably 1-100 mug/mL;
(5) The Rho protein kinase inhibitor is preferably one or more selected from Y27632, hydroxyfasudil and GSK429286A, and the concentration range is preferably 2.5-40 mu M;
(6) The concentration range of dexamethasone is preferably 10-1000 nM;
(7) The concentration range of the neuregulin 1NRG1 is preferably 1-100 ng/mL;
(8) The concentration range of sodium pyruvate is preferably 0.04-25 mM;
(9) The volume concentration of fetal bovine serum FBS relative to the culture medium is preferably 1.25% (v/v) to 20% (v/v);
(10) The concentration range of the fibroblast growth factor bFGF is preferably 1.25-20 ng/mL;
(11) The concentration range of the insulin-like growth factor IGF-1 is preferably 1.25-20 ng/mL;
(12) The volume ratio of the B27 or N2 additive to the culture medium is preferably 1:12.5-1:200;
in an embodiment of the invention, the medium further comprises a starter medium selected from the group consisting of DMEM/F12, DMEM, F12 and RPMI-1640; and one or more antibiotics selected from the group consisting of streptomycin/penicillin, amphotericin B and Primocin.
In a preferred embodiment, the streptomycin concentration is in the range of 25 to 400 μg/mL when the antibiotic is selected from streptomycin/penicillin, the penicillin concentration is in the range of 25 to 400U/mL when the antibiotic is selected from amphotericin B, the concentration is in the range of 0.25 to 4 μg/mL when the antibiotic is selected from Primocin, the concentration is in the range of 25 to 400 μg/mL.
The invention also provides a method for culturing the nasopharyngeal carcinoma organoids. In the method for culturing a nasopharyngeal carcinoma organoid of the present invention, a nasopharyngeal carcinoma primary cell is cultured using the nasopharyngeal carcinoma organoid medium of the present invention.
The nasopharyngeal carcinoma organoid culture method of the invention includes the following steps.
1. And separating a sample from the nasopharyngeal carcinoma solid tumor tissue to obtain the nasopharyngeal carcinoma primary cells. The processing procedure comprises the following steps:
(1) Separating a nasopharyngeal carcinoma tissue sample, adding a basic culture medium and a tissue digestion solution (the addition amount of the tissue digestion solution is 5-15 mL of the tissue digestion solution is used for every 1g of tumor tissue) in a constant-temperature shaking table for digestion, wherein the digestion temperature is 4-37 ℃, the rotation speed of the shaking table is 200-350 rpm, and the digestion time is 3-6 hours;
(2) After digestion, centrifugal separation is carried out, the centrifugal rotation speed is 1200-1600 rpm, the centrifugal time is 2-6 minutes, and the supernatant is discarded after centrifugation.
Wherein the basal medium formulation comprises an initial medium selected from the group consisting of DMEM/F12, DMEM, F12 and RPMI-1640; and one or more antibiotics selected from the group consisting of streptomycin/penicillin, amphotericin B and Primocin. The tissue digestion solution comprises 1640 culture medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNase (50-100U/mL), hyaluronidase (0.5-1 mg/mL), calcium chloride (1-5 mM) and bovine serum albumin BSA (5-10 mg/mL).
2. The nasopharyngeal carcinoma organoid culture medium is prepared, and the primary cells of the nasopharyngeal carcinoma obtained by the steps are cultured.
Re-suspending and counting the primary cells of the nasopharyngeal carcinoma obtained in the step 1 by using a basic culture medium or a nasopharyngeal carcinoma organoid culture medium of the invention, and taking diluted cells with the density of 1-10 multiplied by 10 6 Adding the cell suspension of each/mL into Matrigel matrix gel of equal volume, uniformly mixing, inoculating the mixture into a porous plate, placing the inoculated porous plate into an incubator for 30-60 minutes, and adding nasopharyngeal carcinoma organoid culture medium into the porous plate coated with Matrigel matrix gel for expansion culture after Matrigel is completely coagulated.
The present invention also provides a method for evaluating or screening a drug for treating a nasopharyngeal carcinoma disease, comprising the steps of:
(1) The nasopharyngeal carcinoma organoids are cultivated by using the cultivation method of the nasopharyngeal carcinoma organoids;
(2) Selecting a drug to be detected and diluting according to a required concentration gradient;
(3) Adding the diluted drug to the organoid obtained in (1);
(4) Organoid size or organoid viability testing was performed.
The beneficial effects of the invention include:
(1) The success rate of nasopharyngeal carcinoma organoid culture is improved, and reaches more than 90 percent;
(2) Ensuring that the nasopharyngeal carcinoma organoids which are primarily cultured in vitro can keep the pathological characteristics of patients;
(3) The amplification efficiency is high, the nasopharyngeal carcinoma organoids can be rapidly cultured, and the amplified nasopharyngeal carcinoma organoids can be continuously passed;
(4) The culture cost is controllable, and factors such as expensive Wnt agonists, R-spondin family proteins, noggin proteins, BMP inhibitors and the like are not required to be added into the culture medium;
(5) The nasopharyngeal carcinoma organoids obtained by the culture of the technology are large in quantity, and are suitable for high-throughput screening of candidate compounds and in-vitro sensitivity functional test of high-throughput drugs for patients.
Drawings
FIGS. 1A-1L are graphs showing the effect of additive factors of the nasopharyngeal carcinoma organoids of the present invention on proliferation of nasopharyngeal carcinoma organoids at different concentrations.
FIG. 2A is a photograph of a nasopharyngeal carcinoma organoid obtained after culturing sample NPC22004 for 5 days using a nasopharyngeal carcinoma organoid medium of the present invention under a microscope; fig. 2B is a photograph of a nasopharyngeal carcinoma organoid obtained after culturing a sample NPC220012 for 5 days using a nasopharyngeal carcinoma organoid medium of the present invention, using a microscope.
FIG. 3A is a graph showing the results of pathological and immunohistochemical identification of a nasopharyngeal carcinoma organoid obtained by culturing sample NPC22004 using a nasopharyngeal carcinoma organoid medium of the present invention; fig. 3B is a graph showing the results of pathological and immunohistochemical identification of sample NPC22004 tissue.
FIG. 4A is a photomicrograph of sample NPC22020 after 5 days of nasopharyngeal carcinoma organoid culture using NPC-3 culture medium of this invention; fig. 4B is a photomicrograph of sample NPC22020 after 5 days of nasopharyngeal carcinoma organoid culture with SSL medium.
FIG. 5 shows a comparative histogram of the relative size of the organoids obtained from nasopharyngeal carcinoma organoids cultured on sample NPC22020 using NPC-3 medium and SSL medium, respectively.
FIGS. 6A-6E are graphs showing the results of different drug susceptibility tests of nasopharyngeal carcinoma organoids obtained using the nasopharyngeal carcinoma organoid medium of the present invention.
Detailed Description
For a better understanding of the present invention, the present invention will be further described with reference to the following examples and drawings. The following examples are merely illustrative of the invention and are not intended to be limiting.
Example 1 Effect of various additives in nasopharyngeal carcinoma organoids Medium on proliferation of nasopharyngeal carcinoma organoids
(1) Preparation of nasopharyngeal carcinoma organoid culture medium
First, a basal medium containing the initial medium was prepared. The initial medium may be selected from DMEM/F12, DMEM, F12 or RPMI-1640 as is commonly used in the art. In this example, the basal medium formulation was: DMEM/F12 medium (available from Corning company) +100. Mu.g/mL Primocin (available from InvivoGen company, 0.2% (v/v), commercial product concentration 50 mg/mL).
Different kinds of additives (see table 1) are respectively added into the basic culture medium to prepare the nasopharyngeal carcinoma organoid culture medium containing different additive components.
(2) Isolation and treatment of primary cells of nasopharyngeal carcinoma
1 sample selection
The solid tumor tissue sample of nasopharyngeal carcinoma (intraoperative) is obtained from the patient by a professional medical staff of a professional medical institution, and the patient signs an informed consent. Samples 0.25cm during surgery 3 The tissue preservation solution (manufacturer: miltenyi Biotec) was used for storage and transport.
2 preparation of materials
The surfaces of the 15mL sterile centrifuge tube, the pipette, the 10mL pipette, the sterile gun head and the like are sterilized and then put into an ultra-clean workbench for ultraviolet irradiation for 30 minutes. Basal medium was taken out from the refrigerator at 4℃30 minutes in advance, and tissue digest was taken out from the refrigerator at-20℃30 minutes in advance.
The formula of the tissue digestion liquid comprises the following steps: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg/mL), collagenase IV (2 mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3 mM), bovine serum albumin BSA (10 mg/mL).
Collagenase II, collagenase IV, DNase, hyaluronidase mentioned above were all purchased from Sigma company; calcium chloride was purchased from the division of bioengineering (Shanghai); BSA was purchased from Biofroxx corporation.
3 sample separation
3.1 taking tissue samples in a culture dish in an ultra clean bench, removing the tissue with blood, flushing 2 times with basic culture medium, the tissue was transferred to another petri dish and mechanically separated with a sterile scalpel, dividing tissue blocks into 1X 1mm 3 Size of the material;
3.2 sucking the cut intraoperative tissue into a 15mL centrifuge tube, adding 5mL of basic culture medium, uniformly mixing, and centrifuging at 1500rpm for 4 minutes;
3.3 discarding the supernatant, adding a basic culture medium and a tissue digestion solution in a ratio of 1:3 (note: the addition amount of the tissue digestion solution is about 10mL of tissue digestion solution is used for 1g of tumor tissue), marking the name and the number of a sample, sealing the sample by using a sealing film, and performing digestion in a shaker (known as ZQLY-180N) at 300rpm at 37 ℃, observing whether the digestion is completed every 30 minutes, and judging whether the basis is no macroscopic particles;
3.4 after digestion, undigested tissue mass was filtered off through a 100 μm sieve, and the tissue mass on the sieve was rinsed with basal medium into a centrifuge tube to reduce cell loss, and centrifuged at 1500rpm for 4 minutes at 25 ℃;
3.5 discarding the supernatant, observing whether blood cells exist, if so, adding 8mL of blood cell lysate (purchased from Sigma company), mixing, lysing for 20 minutes at 4 ℃, reversing the mixing for one time, and centrifuging at 1500rpm for 4 minutes at 25 ℃;
3.6 removing the supernatant, adding 2mL of basal medium to re-suspend the cells for later use.
4 cell count and treatment
4.1 looking under the mirror: removing a small amount of resuspended cells, spreading the cells in a culture dish, and observing the density and morphology of cancer cells under a microscope (CNOPTEC, BDS 400);
4.2 viable cell count: after 12. Mu.L of the resuspended cell suspension and 12. Mu.L of trypan blue dye (manufacturer: manufacturing bioengineering (Shanghai) Co., ltd.) were thoroughly mixed, 20. Mu.L was added to a cell counting plate (manufacturer: countstar, specification: 50 pieces/box) and the percentage of viable macro cells (cell size >10 μm) was calculated by a cell counter (Countstar, IC 1000) =viable cell number/total cell number×100%.
(3) Culture of nasopharyngeal carcinoma organoids
The primary cells (NPC 22004, NPC 22006) of nasopharyngeal carcinoma obtained in the above steps are resuspended and counted by using a pre-cooled basic medium, and the cell density is diluted to 1×10 6 400. Mu.L of the diluted cell suspension was removed and added to an equal volume of Matrigel (Corning) and gently mixed, and the mixture was seeded into 96-well plates at 5. Mu.L/well. The inoculated plates were placed in a 37℃cell incubator for 30 minutes until Matrigel was completely coagulated, and then culture media shown in Table 1, which had been restored to room temperature in advance, were added respectively, and the expansion culture was performed by changing the culture media every three days. And taking a picture of the cultured organoids 7 days later, measuring the diameters of the organoids, and comparing the promotion effect of each factor on proliferation of the nasopharyngeal carcinoma organoids. Among them, as experimental controls, a basal medium without any additives was used, and experimental results are shown in table 1.
TABLE 1 additive ingredients in the Medium and organoid proliferation promoting effect
Wherein "+" indicates that the medium to which the additive is added has an effect of promoting proliferation of at least two cases of nasopharyngeal carcinoma organoids isolated from the nasopharyngeal carcinoma tissue, as compared to the basal medium; "-" indicates that the culture medium added with the additive has an effect of inhibiting proliferation on at least one of nasopharyngeal carcinoma organoids isolated from a nasopharyngeal carcinoma tissue; "good" indicates that the culture medium to which the additive was added had no significant effect on proliferation of at least two of the nasopharyngeal carcinoma organoids isolated from the nasopharyngeal carcinoma tissue.
According to the results, factors such as EGF (epidermal growth factor), HGF (hepatocyte growth factor), ITS (ITS cell culture) additive, insulin, Y27632, dexamethasone, neuregulin 1NRG1, sodium pyruvate, FBS (fetal bovine serum), bFGF (fibroblast growth factor), IGF-1, B27 and the like are selected for further culture experiments.
Example 2 proliferation of nasopharyngeal carcinoma organoids by different concentrations of Medium addition factors
Primary nasopharyngeal carcinoma cells were obtained from the intraoperative tissue samples (No. NPC22004, NPC 22006) according to the method of (2) of example 1, and organoid culture was performed using the medium in table 2 below.
TABLE 2 Medium composition (final concentration)
The primary cells of nasopharyngeal carcinoma obtained in the above steps are resuspended and counted by using a pre-chilled basal medium, and the cell density is diluted to 1×10 6 400. Mu.L of the diluted cell suspension was added to an equal volume of Matrigel (Corning) and gently mixed, and the mixture was plated in 96-well plates at 5. Mu.L/well. The inoculated plates were placed in a 37℃cell incubator for 30 minutes until Matrigel was completely coagulated. Media formulated according to the 12 formulations of table 2 were added to each well of a matrigel coated 96-well plate at 200 μl per well.
When the culture medium of the formula 1 is used, 200 mu L of the prepared EGF-containing basic culture medium is added into a 96-well plate inoculated with primary cells on the basis of the formula 1, and the final concentration of EGF is 0.6ng/mL, 1.7ng/mL, 5ng/mL, 15ng/mL and 45ng/mL; and control wells (BC) were set using the medium of formula 1. The final concentration of other additive factors in the series of media was the same as that of NPC-3 media. Experiments of the following formulations 2 to 12 were also performed in the same manner and will not be described again.
When the culture medium of the formula 2 is used, 200 mu L of prepared basal culture medium containing HGF is respectively added into a 96-well plate inoculated with primary cells on the basis of the formula 2, and the final concentration of HGF is respectively 1ng/mL, 3ng/mL, 10ng/mL, 30ng/mL and 100ng/mL; and control wells (BC) were set using the medium of formula 2.
When the culture medium of the formula 3 is used, 200 mu L of prepared basal culture medium containing ITS is respectively added into a 96-well plate inoculated with primary cells on the basis of the formula 3, and the final volume ratio of ITS to the culture medium is respectively 1:25, 1:50, 1:100, 1:200 and 1:400; and control wells (BC) were set using the medium of formula 3.
When the culture medium of the formula 4 is used, 200 mu L of the prepared basal medium containing insulin is added to each well of a 96-well plate inoculated with primary cells on the basis of the formula 4, and the final concentration of insulin is 1 mu g/mL, 3 mu g/mL, 10 mu g/mL, 30 mu g/mL and 100 mu g/mL respectively; and control wells (BC) were set using the medium of formula 4.
When the culture medium of formula 5 is used, 200 mu L of the prepared basal culture medium containing Y27632 is added to a 96-well plate inoculated with primary cells on the basis of formula 5, and the final concentration of Y27632 is 2.5 mu M, 5 mu M, 10 mu M, 20 mu M and 40 mu M; and control wells (BC) were set using the medium of formula 5.
When the culture medium of the formula 6 is used, 200 mu L of the prepared basal culture medium containing dexamethasone is respectively added into a 96-well plate inoculated with primary cells on the basis of the formula 6, and the final concentration of the dexamethasone is respectively 10nM, 30nM, 100nM, 300nM and 1000nM; and control wells (BC) were set using the medium of formula 6.
When the culture medium of the formula 7 is used, 200 mu L of prepared basal culture medium containing NRG1 is respectively added into a 96-well plate inoculated with primary cells on the basis of the formula 7, and the final concentration of the NRG1 is respectively 1ng/mL, 3ng/mL, 10ng/mL, 30ng/mL and 100ng/mL; and control wells (BC) were set using the medium of formula 7.
When the culture medium of the formula 8 is used, 200 mu L of the prepared basal culture medium containing sodium pyruvate is added to each well of a 96-well plate inoculated with primary cells on the basis of the formula 8, and the final concentration of the sodium pyruvate is 0.04mM, 0.2mM, 1mM, 5mM and 25mM respectively; and control wells (BC) were set using the medium of formula 8.
When the culture medium of formula 9 is used, 200 mu L of the prepared basal medium containing FBS is added to each well of a 96-well plate inoculated with primary cells on the basis of formula 9, and the final volume concentration of FBS is 1.25% (v/v), 2.5% (v/v), 5% (v/v), 10% (v/v) and 20% (v/v) respectively; and control wells (BC) were set using the medium of formula 9.
When the culture medium of the formula 10 is used, 200 mu L of prepared basic culture medium containing bFGF is respectively added into a 96-well plate inoculated with primary cells on the basis of the formula 10, and the final concentration of the bFGF is respectively 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL; and control wells (BC) were set using the medium of formulation 10.
When the culture medium of formula 11 is used, 200 mu L of the prepared IGF-1-containing basal culture medium per well is added to a 96-well plate inoculated with primary cells on the basis of formula 11, and the final concentration of IGF-1 is 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL, respectively; and control wells (BC) were set using the medium of formula 11.
When the culture medium of formula 12 is used, 200 mu L of prepared basal culture medium containing B27 is respectively added into a 96-well plate inoculated with primary cells on the basis of formula 12, and the final volume ratio of B27 to the culture medium is respectively 1:12.5, 1:25, 1:50, 1:100 and 1:200; and control wells (BC) were set using the medium of formula 12.
And taking a picture of the cultured organoids 7 days later, measuring the diameters of the organoids, and comparing the promotion effect of the concentrations of the factors on proliferation of the nasopharyngeal carcinoma organoids. The data collected from 2 samples are summarized in FIGS. 1A-1L. In FIGS. 1A-1L, the multiplication factor on the ordinate is the ratio of the diameter of the organoid obtained by 7 days of incubation with each medium to the diameter of the organoid obtained by 7 days of incubation with the corresponding BC control well. The ratio of more than 1 indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is better than that of a control Kong Peiyang base; the ratio is smaller than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is weaker than that of the culture medium with the control holes.
According to the results of FIGS. 1A to 1L, the EGF content is preferably 0.6 to 45ng/mL, more preferably 1.7 to 15ng/mL; the HGF content is preferably 1 to 100ng/mL, more preferably 10 to 30ng/mL; the volume ratio of ITS to the culture medium is preferably 1:25-1:400, more preferably 1:50-1:200; the content of insulin is preferably 1 to 100. Mu.g/mL, more preferably 3 to 30. Mu.g/mL; the content of Y27632 is preferably 2.5 to 40. Mu.M, more preferably 5 to 20. Mu.M; the dexamethasone content is preferably 10-1000 nM, more preferably 30-300 nM; the NRG1 content is preferably 1 to 100ng/mL, more preferably 3 to 30ng/mL; the content of sodium pyruvate is preferably 0.04 to 25mM, more preferably 0.2 to 5mM; the volume concentration of FBS is preferably 1.25% (v/v) to 20% (v/v), more preferably 2.5% (v/v) to 10% (v/v); the bFGF content is preferably 1.25 to 20ng/mL, more preferably 2.5 to 10ng/mL; the IGF-1 content is preferably 1.25-20 ng/mL, more preferably 2.5-20 ng/mL; the volume ratio of B27 to the culture medium is preferably 1:12.5 to 1:200.
Example 3 cultivation and identification of nasopharyngeal carcinoma organoids
Primary cells (NPC 22004, NPC 22012) of nasopharyngeal carcinoma obtained as described in (2) of example 1 were resuspended and counted with the nasopharyngeal carcinoma organoid medium NPC-3 of the present invention, and the cell density was diluted to 1X 10 6 400. Mu.L of the diluted cell suspension was removed at each mL, added to an equal volume of Matrigel (Corning) and gently mixed, and the mixture was seeded at 50. Mu.L/well in a 24-well plate. Placing the inoculated culture plate into a cell culture box at 37 ℃ for 30 minutes, adding NPC-3 which is restored to room temperature in advance into the cell culture box after Matrigel is completely solidified, and performing expansion culture by replacing the culture medium every three days every 500 mu L of the culture plate.
On day 5, the cultured nasopharyngeal carcinoma organoids were observed using a microscope (EVOS M500 of Invitrogen). FIGS. 2A and 2B are photographs of nasopharyngeal carcinoma organoids obtained after culturing of sample NPC22004, NPC22012, respectively, under a 10-fold objective lens. The nasopharyngeal carcinoma organoids are in regular spherical shape under the mirror, and the surface is smoother.
And carrying out pathological and immunohistochemical identification on the cultured nasopharyngeal carcinoma organoids, and simultaneously carrying out pathological and immunohistochemical identification on corresponding tissue samples, and comparing the consistency of organoids and tissue results.
Fig. 3A is a photograph taken at 20 x magnification under an objective lens, and the result of pathological and immunohistochemical identification of the nasopharyngeal carcinoma organoid obtained after in vitro culture of sample NPC22004 using the nasopharyngeal carcinoma organoid medium of the present invention. As shown in fig. 3A, HE results show that the structural morphology of organoids is that of cancerous tissue; the nasopharyngeal carcinoma markers P40, CK5/6, P53 and Ki67 are expressed in high, so that the sample is indicated to be nasopharyngeal carcinoma. Fig. 3B is a photograph taken at 20 x magnification with an objective lens, and the pathological and immunohistochemical identification of NPC22004 corresponding tissues was performed. Comparing FIGS. 3A and 3B, the results show that the nasopharyngeal carcinoma organoids cultured using the medium NPC-3 of the present invention are consistent with the diagnosis results of nasopharyngeal carcinoma tissue.
Example 4 comparison of the culture Effect with the existing Medium
(1) Preparation of control Medium
Medium used in formulation literature (Sasidharan Swarnalatha Lucky et al, front in oncology.2021,23; 11:622244) was formulated as DMEM/F12 medium (from Corning Inc.) +4μg/mL Heparin (from MCE Inc.) +125ng/mL Hydrocortisone (from MCE Inc.) +50ng/mL epidermal growth factor (from R & D Inc.) +50ng/mL bFGF (from sin biological Inc.) +20ng/mL fibroblast growth factor 10 (from sin biological Inc.) +10 μM SB202190 (from MCE Inc. +500nM A8301 (from MCE Inc.) +10 μ M Y27632 (from MCE Inc.) +250ng/mL amphotericin B (from Selleck Inc.) +10 μg/mL gentamicin (from MCE Inc.) +2mM GlutaMAX (from nin Inc.). Hereinafter, this is abbreviated as SSL medium.
(2) Nasopharyngeal carcinoma organoid culture
Primary nasopharyngeal carcinoma cells were obtained from the intraoperative tissue sample NPC22020 as in (2) of example 1, and organoid culture was performed as in example 3 using NPC-3 medium and SSL medium, respectively.
On day 5 of culture, the cultured nasopharyngeal carcinoma organoids were observed using a microscope (EVOS M500 of Invitrogen). FIGS. 4A and 4B are photographs taken under a 10-fold objective lens of organoids cultured in NPC-3 medium and SSL medium, respectively, and FIG. 5 is a comparative bar graph of the relative sizes of organoids cultured in both media.
From the results of FIGS. 4A, 4B and 5, it can be seen that NPC-3 medium significantly promotes the amplification and culture of nasopharyngeal carcinoma organoids as compared to SSL medium.
Example 5 nasopharyngeal carcinoma organoids amplified using the culture medium of the present invention for drug screening
(1) Nasopharyngeal carcinoma organoid culture
Primary cells of nasopharyngeal carcinoma were isolated from a sample (NPC 22039) in a nasopharyngeal carcinoma surgery as in (2) of example 1, and organoid cultured using NPC-3 medium, and drug screening was performed when the diameter of the nasopharyngeal carcinoma organoid exceeded 50 μm.
(2) Screening pharmaceutical formulations
5 drugs (paclitaxel, docetaxel, cisplatin, carboplatin, 5-Fu; all available from MCE company) were prepared at 5 concentration gradients according to the following table and stored for use.
TABLE 3 preparation of paclitaxel, docetaxel, cisplatin, carboplatin, 5-Fu drug additive solutions
| Preparation of concentration (mM) | Paclitaxel (Taxol) | Docetaxel (docetaxel) | Cisplatin (cisplatin) | Carboplatin | 5-Fu |
| 1x Cmax | 8 | 5 | 20 | 14 | 0.7 |
| 0.5x Cmax | 4 | 2.5 | 10 | 7 | 0.35 |
| 0.25x Cmax | 2 | 1.25 | 5 | 3.5 | 0.18 |
| 0.125x Cmax | 1 | 0.63 | 2.5 | 1.75 | 0.09 |
| 0.06x Cmax | 0.5 | 0.31 | 1.25 | 0.88 | 0.04 |
(3) Dosing
Taking out the prepared medicine, placing at room temperature, diluting the medicine with NPC-3 culture medium for 1000 times for standby. Taking out the organoids obtained by culturing in the step (1) from the incubator, removing the culture medium in the culture well, and slowly introducing the culture medium containing the drug into the culture well along the wall of the well. After the addition, the surface of the 96-well plate is sterilized and then transferred to a 37 ℃ cell incubator for continuous culture, and the organoid activity is measured after 6 days.
(4) Organoid vitality test
Taking out CellTiter-Glo luminescent reagent (purchased from Promega company) in a refrigerator at 4 ℃, taking 10ml of reagent into a sample adding groove, taking out a 96-well plate to be detected from an incubator, adding 20 mu LCellTiter-Glo luminescent reagent into each well, standing for 10 minutes, uniformly mixing, and detecting by using a multifunctional enzyme-labeled instrument (Perkin Elmer company Envision).
(5) Data processing
Drug inhibition (%) = 100% - (day 6 culture well chemiluminescence values according to the formula Drug treatment group Chemiluminescent number of culture well on day 6 Control group ) 100% inhibition of the different drugs was calculated and the results are shown in FIGS. 6A-6E. Fig. 6A (paclitaxel), fig. 6B (docetaxel), fig. 6C (cisplatin), fig. 6D (carboplatin) and fig. 6E (5-Fu) are graphs of inhibition of nasopharyngeal carcinoma organoids at different concentrations of the test drug. As can be seen from fig. 6A-6E, the data error values for the drug treatment groups were small, indicating that the data remained substantially identical between the same drug wells when drug screening was performed using the methods of the present invention. Of the five antitumor drugs, paclitaxel had some inhibitory effect on the growth of NPC22039 organoids in this sample, docetaxel, cisplatin, carboplatin and 5-Fu were not substantially able to inhibit the growth of NPC22039 organoids, indicating that the organoids of the same patient were differently sensitive to different drugs. According to the result, the effectiveness and the effective dosage of the nasopharyngeal carcinoma patient in clinical use of the medicine can be judged.
Industrial applicability
The invention provides a culture medium and a culture method for culturing nasopharyngeal carcinoma organoids, which can be used for evaluating and screening curative effects of medicines by using the organoids obtained by culture. Thus, the present invention is suitable for industrial applications.
Although the present invention has been described in detail herein, the present invention is not limited thereto, and modifications may be made by those skilled in the art in light of the present principles, and it is therefore intended that all such modifications as fall within the scope of the present invention.
Claims (7)
1. A nasopharyngeal carcinoma organoid medium comprising:
epidermal growth factor, hepatocyte growth factor, ITS cell culture additive, insulin, rho protease inhibitor, dexamethasone, neuregulin 1, sodium pyruvate, fetal bovine serum, fibroblast growth factor, insulin-like growth factor number one, and at least one cell culture additive selected from N2 and B27.
2. The nasopharyngeal carcinoma organoid medium of claim 1, wherein the content of each component of said medium satisfies any one or more or all of the following:
the concentration range of the epidermal cell growth factor is 0.6-45 ng/mL;
the concentration range of the hepatocyte growth factor is 1-100 ng/mL;
the volume ratio of the ITS cell culture additive to the culture medium is 1:25-1:400;
the concentration range of the insulin is 1-100 mug/mL;
the Rho protein kinase inhibitor is one or more selected from Y27632, hydroxyfasudil and GSK429286A, and the concentration range is 2.5-40 mu M;
the concentration range of the dexamethasone is 10-1000 nM;
the concentration range of the neuregulin 1 is 1-100 ng/mL;
the concentration range of the sodium pyruvate is 0.04-25 mM;
the volume concentration of the fetal bovine serum relative to the culture medium is 1.25% (v/v) to 20% (v/v);
the concentration range of the fibroblast growth factor is 1.25-20 ng/mL;
the concentration range of the insulin-like growth factor I is 1.25-20 ng/mL;
the volume ratio of the B27 or N2 additive to the culture medium is 1:12.5-1:200.
3. The nasopharyngeal carcinoma organoid medium according to claim 1 or 2, wherein said medium further comprises a primary medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from the group consisting of streptomycin/penicillin, amphotericin B and Primocin.
4. The nasopharyngeal carcinoma organoid medium of claim 1 or 2, wherein said medium is free of Wnt agonist, R-spondin family protein, noggin protein, or BMP inhibitor.
5. A method for culturing nasopharyngeal carcinoma organoids, which is characterized by comprising the following steps:
culturing a primary cell of nasopharyngeal carcinoma using the nasopharyngeal carcinoma organoid medium according to any one of claims 1-4.
6. The culture method according to claim 5, comprising the steps of:
(1) Separating a sample from a solid tumor tissue of the nasopharyngeal carcinoma to obtain primary cells of the nasopharyngeal carcinoma;
(2) Preparing a nasopharyngeal carcinoma organoid medium according to any one of claims 1-4, and performing organoid culture on the nasopharyngeal carcinoma primary cells obtained in step (1).
7. A method for evaluating or screening a drug for treating a nasopharyngeal carcinoma disease, comprising the steps of:
(1) Culturing a nasopharyngeal carcinoma organoid using the method for culturing a nasopharyngeal carcinoma organoid of claim 5 or 6;
(2) Selecting a drug to be detected and diluting according to a required concentration gradient;
(3) Adding the diluted drug to the organoid obtained in (1);
(4) And performing organoid size or organoid activity detection.
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| PCT/CN2022/125112 WO2024065883A1 (en) | 2022-09-28 | 2022-10-13 | Culture medium for nasopharyngeal carcinoma organoid culture, and culture method and use thereof |
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| CN109679915B (en) * | 2019-02-27 | 2021-12-31 | 南方医科大学南方医院 | Culture and identification method of nasopharyngeal carcinoma organoid |
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