CN113278586A - Culture medium and culture method for culturing thyroid cancer organoid - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention provides a culture medium for thyroid cancer organoids and a culture method thereof. The culture medium contains a basal medium, B27, a p38MAPK inhibitor and thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01. The organoid culture by adopting the culture medium has low cost, low operation difficulty, high culture success rate and more passage times.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a culture medium and a culture method for culturing thyroid cancer organoids.
Background
Thyroid cancer is a malignant tumor originated from thyroid follicular epithelium or perifollicular epithelial cells, is a common malignant tumor of the head and neck, and poses serious threats to the physical health and the quality of life of patients. Thyroid cancer can be classified into: papillary Thyroid Carcinoma (PTC), Follicular Thyroid Carcinoma (FTC), Medullary Thyroid Carcinoma (MTC), and Anaplastic Thyroid Carcinoma (ATC). PTC and FTC are collectively called differentiated thyroid cancer, with PTC accounting for approximately 85% to 90% of all thyroid cancers. While most differentiated thyroid cancers are alleviated by surgery, post-operative radioiodine-131, and thyroid hormone suppression therapy, some patients exhibit radioiodine refractory states in their natural course or after treatment. The survival rate of thyroid cancer patients with radioactive iodine refractory property, metastatic property or advanced stage is obviously reduced, and the thyroid cancer patients are difficult points and hot points of the current clinical diagnosis and treatment of the thyroid cancer.
Cancer treatment has turned into targeted individualized precision medicine based on tumor mutation profiling. Clinical tests and Meta analysis show that the molecular targeted drug has wide prospect on the curative effect of refractory thyroid cancer. At present, the individualized precise medical treatment of tumors lacks a proper preclinical evaluation model. The traditional 2D cultured cell line is simple to operate and suitable for screening high-flux antitumor drugs, but lacks the interaction between cells and cell matrixes, and cannot reproduce the diversity characteristics of tumors. In addition, cell lines lose tumor heterogeneity during long-term culture in vitro, cannot represent the genotype and pathological features of the parent tumor, and cannot mimic the complex structure of the tumor and intercellular signaling pathways in the patient. Although patient-derived xenograft models can mimic the characteristics of parental tumors, maintain tumor heterogeneity and genomic stability, they are inefficient to construct, long-lasting, expensive, species-diverse, and unsuitable for high-throughput drug screening. In recent years, the emergence and development of organoid culture technology brings a brand new tumor model for us. Organoids are three-dimensional cell complexes that are structurally and functionally similar to a target organ or tissue, induced by in vitro 3D culture techniques to differentiate stem cells or organ progenitor cells, have stable phenotypic and genetic characteristics, and can be cultured in vitro for long periods of time. Compared with the traditional 2D cell culture and animal models, the in vitro model with the 3D structure can not only show the cell structure and behavior of the source organ, maintain the stability of gene expression, but also accurately predict the reaction of a patient to an anti-tumor drug, and is a potential substitute for human pathological tissues.
In 2009, the Hans Clevers group first developed the universal culture conditions for organoids, i.e. adding epidermal growth factor, Wnt signal agonist R-spondin-1 and bone morphogenetic protein signal inhibitor Noggin into the culture medium, and this culture system also prepared the first organoid model, small intestine organoid (Sato et al, Nature,2009,459(7244): 262-.
In 2011, the Hans Clevers team developed organoid culture systems, and successfully cultured multiple organoid disease models of small intestine and colon capable of long-term growth in vitro by introducing nicotinamide, p38MAPK inhibitor, gastrin, a83-01, fibroblast growth factor, etc. (Sato et al, Gastroenterology,2011,141(5): 1762-.
Organoid models of various types of tumors have been reported in succession by several groups at home and abroad in recent years, but the factors added to these tumor organoid media are mostly based on the media components originally designed by the Hans Clevers group. At present, only 1 research on thyroid cancer organoids is reported at home and abroad (Sondorp et al, cancers (Basel),2020,12(11):3212), and the components of a culture medium of the research are conventional components for culturing tumor organoids. The culture medium has more added factors and high cost, and the thyroid cancer organoid model which is stably subcultured for a long time is difficult to obtain in vitro.
Therefore, the development of a culture medium and a culture method for efficiently obtaining thyroid cancer organoids at low cost is urgently needed.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium and a culture method for thyroid cancer organoids and application of the culture medium.
In a first aspect, the invention provides a culture medium for a thyroid cancer organoid.
A culture medium for a thyroid cancer organoid, the culture medium comprising basal medium, B27, a p38MAPK inhibitor, and thyroid stimulating hormone.
The medium may be free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01. In some embodiments, the medium does not contain any of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01.
The p38MAPK inhibitor may include at least one selected from SB202190, SB203580, and Doramapimod; alternatively, the p38MAPK inhibitor is Doramapimod.
The basal medium can be Advanced DMEM/F12 medium.
The medium may further comprise an epidermal growth factor.
The concentration of the p38MAPK inhibitor in the culture medium may be 1-20. mu.M based on the total volume of the culture medium. In some embodiments, the concentration of the p38MAPK inhibitor in the culture medium is 5-15 μ M, based on the total volume of the culture medium. In some embodiments, the concentration of the p38MAPK inhibitor in the culture medium is 8-12 μ M, based on the total volume of the culture medium. In some embodiments, the concentration of the p38MAPK inhibitor in the culture medium is 10 μ M, based on the total volume of the culture medium.
The volume percentage concentration of the B27 in the culture medium can be 1-5% based on the total volume of the culture medium. In some embodiments, the percentage concentration by volume of B27 in the medium is between 2% and 4% based on the total volume of the medium. In some embodiments, the percentage concentration by volume of B27 in the medium is 2%, based on the total volume of the medium.
The B27 can be B27 supplement 50 x.
The concentration of the thyroid stimulating hormone in the medium may be 1-50ng/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 10-40ng/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 20-30ng/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 20ng/mL based on the total volume of the medium.
The concentration of the epidermal growth factor in the culture medium may be 1-100ng/mL based on the total volume of the culture medium. In some embodiments, the epidermal growth factor is present in the culture medium at a concentration of 20-80ng/mL based on the total volume of the culture medium. In some embodiments, the epidermal growth factor is present in the culture medium at a concentration of 30-70ng/mL based on the total volume of the culture medium. In some embodiments, the epidermal growth factor is present in the culture medium at a concentration of 40-60ng/mL based on the total volume of the culture medium. In some embodiments, the epidermal growth factor is present in the culture medium at a concentration of 50ng/mL based on the total volume of the culture medium.
The medium may further comprise at least one of N-acetylcysteine and nicotinamide.
In the culture medium, the concentration of the N-acetylcysteine is 1-5mM based on the total volume of the culture medium. In some embodiments, the concentration of N-acetylcysteine in the medium is 1-4mM, based on the total volume of the medium. In some embodiments, the concentration of N-acetylcysteine in the medium is 2-3mM, based on the total volume of the medium. In some embodiments, the concentration of N-acetylcysteine in the medium is 2mM based on the total volume of the medium.
In said medium, said nicotinamide is present at a concentration of 1-20mM based on the total volume of said medium. In some embodiments, said nicotinamide is present in said medium at a concentration of 5-20mM, based on the total volume of said medium. In some embodiments, said nicotinamide is present in said medium at a concentration of 10-15mM, based on the total volume of said medium. In some embodiments, the concentration of nicotinamide in said medium is 10mM, based on the total volume of said medium.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising basal medium, B27, p38MAPK inhibitor, and thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01; the p38MAPK inhibitor includes at least one selected from SB202190, SB203580, and Doramapimod.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising basal medium, B27, p38MAPK, and thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01; the p38MAPK inhibitor is Doramapimod.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising a basal medium, an epidermal growth factor, B27, a p38MAPK inhibitor, and a thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising a basal medium, an epidermal growth factor, B27, a p38MAPK inhibitor, and a thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01; the p38MAPK inhibitor is Doramapimod.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising basal medium, B27, p38MAPK inhibitor, and thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01; the medium further comprises at least one of N-acetylcysteine and nicotinamide.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising a basal medium, an epidermal growth factor, B27, a p38MAPK inhibitor, and a thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01; the p38MAPK inhibitor includes at least one selected from SB202190, SB203580, and Doramapimod; the medium further comprises at least one of N-acetylcysteine and nicotinamide.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising a basal medium, an epidermal growth factor, B27, a p38MAPK inhibitor, and a thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01; the p38MAPK inhibitor is Doramapimod; the medium further comprises at least one of N-acetylcysteine and nicotinamide.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium comprising a basal medium, an epidermal growth factor, B27, a p38MAPK inhibitor, and a thyroid stimulating hormone; and the culture medium does not contain any one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor and A83-01; the p38MAPK inhibitor is Doramapimod; the medium further comprises at least one of N-acetylcysteine and nicotinamide.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid, the culture medium consisting of basal medium, B27, Doramapimod, and thyroid stimulating hormone.
In a second aspect, the invention provides the use of a culture medium as described above.
Use of a medium according to the first aspect for culturing a thyroid cancer organoid; the culture medium is adopted for culture, the cost is low, the operation difficulty is low, the culture success rate is high, the number of passages is large, and the obtained thyroid cancer organoid is large in size and high in growth speed.
In a third aspect, the present invention provides a culture method for culturing thyroid cancer organoids using the aforementioned medium.
A culture method for culturing thyroid cancer organoids using the medium according to the first aspect, comprising the steps of:
(1) taking thyroid cancer tissues, cleaning, cutting into pieces, and digesting with digestive enzyme;
(2) terminating digestion; centrifuging, discarding the supernatant to obtain a first precipitate, re-suspending the first precipitate, filtering, centrifuging the filtrate, discarding the supernatant to obtain a second precipitate, re-suspending the second precipitate with an Advanced DMEM/F12 culture medium and matrigel, inoculating, and incubating;
(3) adding the culture medium to culture to obtain the thyroid cancer organoid.
The digestive enzymes include collagenase type II and/or pancreatin substitutes.
The volume ratio of the matrigel to the Advanced DMEM/F12 culture medium is 3:1-10: 1.
In some embodiments of the present invention, a culture method for culturing a thyroid cancer organoid using the medium according to the first aspect comprises the steps of:
(1) taking thyroid cancer tissues, cleaning, cutting into pieces, and digesting with type II collagenase at 37 ℃ for 1 hour;
(2) adding Hanks liquid to stop digestion; centrifuging, discarding supernatant to obtain a first precipitate, re-suspending the first precipitate with Hanks liquid, filtering with a filter screen of 70 μm, centrifuging filtrate, discarding supernatant to obtain a second precipitate, adding Advanced DMEM/F12 culture medium to mix with the second precipitate, adding matrigel for re-suspension, wherein the volume ratio of the matrigel to the Advanced DMEM/F12 culture medium is 3:1-10:1, inoculating, and incubating;
(3) adding the culture medium, culturing at 37 deg.C for 7-14 days, and replacing the culture medium every 3-4 days to obtain the thyroid cancer organoid.
Advantageous effects
Compared with the prior art, the invention has the following beneficial effects:
1. thyroid stimulating hormone is added into the culture medium, so that the culture success rate and the passage times of thyroid cancer organoids can be improved.
2. The culture medium does not contain Wnt3a, R-spondin-1, noggin, fibroblast growth factor and A83-01 which are commonly used in the prior art, only adopts B27, p38MAPK inhibitor and thyroid stimulating hormone, and rather, has the unexpected technical effects of better culturing thyroid cancer organoids and greatly reducing cost and operation difficulty.
3. According to the growth characteristics of thyroid cancer source cells, key growth factors and inhibitory factors in various signal paths are screened and prepared according to a certain proportion, the content of each factor in the prepared culture medium is appropriate, thyroid cancer cells can effectively form organoid in a 3D environment, the culture success rate can reach at least 84%, and the number of passages can reach at least 10.
4. The thyroid cancer organoid model can be quickly built in vitro (1-2 weeks), can be used for radioiodine pretreatment test and drug sensitivity test, and provides effective guidance for clinical treatment of patients.
5. There is currently no suitable in vitro model for clinical treatment of thyroid cancer. The thyroid cancer organoid obtained by the invention can meet the requirements of scientific research, and can become a novel and effective clinical precursor external model in the aspects of predicting the clinical radioiodine treatment of thyroid cancer and guiding clinical medication.
6. The culture medium and the culture method can realize the rapid amplification of cells from thyroid cancer tissues of patients to form organoids by reducing the cost of the culture medium and simplifying the culture process on the premise of ensuring the success rate and the stable growth of the thyroid cancer organoids. The invention is beneficial to the large-scale production of thyroid cancer organoids for scientific research and preclinical radioiodine sensitivity and drug screening tests, and has important significance for promoting the development of individualized precise medical treatment of thyroid cancer.
Drawings
FIG. 1 is a photomicrograph of thyroid cancer organoids cultured in medium of formulas 1-8 of example 3; in the figure, Nac represents N-acetylcysteine; SB denotes SB 202190; EGF represents an epidermal growth factor; nic represents nicotinamide; RSPO represents R-spondin-1 recombinant protein; noggin represents Noggin; FGF-7 represents fibroblast growth factor-7; FGF-10 denotes fibroblast growth factor-10.
FIG. 2 is a photomicrograph of thyroid cancer organoids cultured in medium of formula 9 through formula 14 from example 3; in the figure, Nac represents N-acetylcysteine; SB denotes SB 202190; EGF represents an epidermal growth factor; TSH denotes thyroid stimulating hormone; dor denotes Doramapimod.
FIG. 3 is a hematoxylin-eosin staining and immunohistochemical staining examination visualization of thyroid cancer organoids and corresponding cancer tissues cultured in medium of formula 11 in example 3; in the figure, H & E represents hematoxylin-eosin staining; galectin-3 represents an immunohistochemical staining of Galectin-3; CK19 represents immunohistochemical staining of keratin 19.
Description of the terms
In the present invention, rpm means revolutions per minute; μ M means micromoles per liter; nM represents nanomoles per liter; μ g means μ g; μ L means μ L; ng/mL represents nanograms per milliliter; mL means mL; g at centrifugation is a centrifugal acceleration unit, for example, 200g represents a centrifugal acceleration of 200 times a gravitational acceleration; U/mL represents "units per milliliter".
In the present invention, the Doramapimod is a p38MAPK inhibitor with CAS number 285983-48-4; SB202190 is a p38MAPK inhibitor having CAS number 152121-30-7; the SB203580 is a p38MAPK inhibitor with the CAS number of 152121-47-6; the Noggin represents Noggin.
In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, some non-limiting examples are further disclosed below, and the present invention is further described in detail.
The reagents used in the present invention are either commercially available or can be prepared by the methods described herein.
Reagents, equipment and sources thereof used in the present invention:
the following examples are commercially available in major equipment unless otherwise specified.
The main apparatus is as follows: a cell culture box (manufacturer: Thermo), an inverted microscope (manufacturer: Leica), a biological safety cabinet (manufacturer: hongkongkang biomedical science and technology control corporation), a low-speed desktop centrifuge (manufacturer: Eppendorf), a constant-temperature water bath (manufacturer: shanghai-heng scientific instruments corporation), a paraffin slicer (manufacturer: Thermo), a pipette (manufacturer: Eppendorf), and the like.
Example 1: culture media of different compositions
The media were prepared according to the formula shown in Table 1.
Table 1: culture medium formula with different components
Example 2: culture media of different compositions
The media were prepared according to the formula shown in Table 2.
Table 2: culture medium formula with different components
Example 3: culture of thyroid cancer organoids
The operation is as follows: the following procedures were carried out on thyroid cancer organoids obtained from patients clinically using the culture media of different formulations described in example 1 and example 2, respectively:
(1) cleaning thyroid cancer tissue, and cutting into pieces of about 1mm3The pieces of (a) were digested with collagenase type II at 37 ℃ for 1 hour;
(2) adding Hanks liquid to stop digestion; centrifuging, discarding supernatant to obtain a first precipitate, re-suspending the first precipitate by Hanks liquid, filtering by a 70-micron cell filter screen, counting cells, centrifuging filtrate, discarding supernatant to obtain a second precipitate, mixing the second precipitate with Advanced DMEM/F12 culture medium, adding matrigel for re-suspending, wherein the volume ratio of the matrigel to the Advanced DMEM/F12 culture medium is 3:1, inoculating, incubating for 5 minutes, and then inverting for 10 minutes to solidify the matrigel;
(3) adding the culture medium, culturing at 37 ℃ for 14 days, and replacing the culture medium every 3 days to obtain the thyroid cancer organoid.
And carrying out microscopic bright field observation, hematoxylin-eosin staining and immunohistochemical staining detection on the obtained thyroid cancer organoids.
As a result: the thyroid cancer organoids obtained using the medium described in example 1 are shown in FIG. 1; the thyroid cancer organoids obtained using the medium described in example 2 are shown in FIG. 2; hematoxylin-eosin staining and immunohistochemical staining detection of thyroid cancer organoids and corresponding thyroid cancer tissues cultured in the medium of formula 11 are shown in fig. 3.
And (4) analyzing results:
(1) as can be seen from FIG. 1, the thyroid cancer organoids obtained by culturing the cells with the culture media described in formulas 5 to 8 have small size and slow growth rate, while those obtained by culturing the cells with the culture medium of formula 4 containing only 4 factors, namely B27, N-acetylcysteine, epidermal growth factor and SB202190, have large size and fast growth rate, which proves that the 4 factors, namely B27, N-acetylcysteine, epidermal growth factor and SB202190, are not indispensable for successful culture of the thyroid cancer organoids without adding other factors.
(2) As can be seen from the morphological diagrams of thyroid cancer organoids cultured according to formulas 1 to 4 in FIG. 1, thyroid organoids with large volume and high growth rate can be successfully cultured in a culture medium containing only 4 factors, namely B27, N-acetylcysteine, epidermal growth factor and SB202190, and the addition of other factors cannot bring more beneficial technical effects on the growth of thyroid cancer organoids.
(3) As can be seen from FIG. 2, in terms of the growth rate of thyroid cancer organoids, the medium of formula 11 containing only B27, thyroid stimulating hormone and Doramapimod > formula 10 containing only B27, N-acetylcysteine, epidermal growth factor, SB202190, thyroid stimulating hormone and Doramapimod; it was demonstrated that the growth rate of thyroid cancer organoids could be greatly increased by using a medium containing only B27, thyroid stimulating hormone and Doramapimod, whereas the growth rate could be decreased by the addition of other factors.
(4) As can be seen from FIG. 2, any of the components "B27, thyroid stimulating hormone and Doramapimod" had a significant effect on increasing the growth rate of thyroid cancer organoids, with the highest growth rate when cultured in medium containing B27, thyroid stimulating hormone and Doramapimod.
(5) As can be seen from the formula 9 in FIG. 2 and the formula 11 in FIG. 2, the culture medium containing B27, thyroid stimulating hormone and Doramapimod has a remarkable technical effect in that the culture medium containing B27, N-acetylcysteine, epidermal growth factor and SB202190 is more beneficial to the culture of thyroid cancer organoids than the culture medium containing B27, N-acetylcysteine, epidermal growth factor and SB202190 in the prior art.
(6) As can be seen in FIG. 3, the resulting thyroid cancer organoids were capable of reducing the histological and marker protein expression characteristics of the parent tumor by culturing in a medium containing B27, thyroid stimulating hormone, and Doramapimod.
Example 4: culture success rate of different culture media
Thyroid cancer tissues of different patients were taken, and the formulations 1 to 4 in example 1 and 10 to 11 in example 2 were taken, and cultured for 25 times by the method described in example 3, and the culture success rates are shown in table 3.
Table 3: culture success rate of different culture media
| Culture medium | Number of successful cultivations | Success rate of cultivation |
| Formulation 1 | 17 | 68％ |
| Formulation 2 | 18 | 72 |
| Formulation | ||
| 3 | 19 | 76％ |
| Formulation 4 | 18 | 72％ |
| Formulation 10 | 21 | 84％ |
| Formulation 11 | 23 | 92％ |
Example 5: organoid passage
First, passage of organoids
Reagent:
and (3) second digestive juice: pancreatin substitute solution containing 10. mu.M of Y-27632.
Culture medium: any of the formulations 1 to 4 of example 1 and 10 to 11 of example 2 (the medium used for subculture was the same as that used for organoids).
The operation is as follows:
centrifuging the thyroid cancer organoids obtained in example 3 at 4 ℃ for 5 minutes at 200g, discarding the supernatant to obtain a fourth precipitate, adding the second digestion solution to the fourth precipitate, and digesting the fourth precipitate for 5 minutes in a shaker at 37 ℃ and a rotation speed of 120 rpm; adding Advanced DMEM/F12 medium containing 20% by volume fetal bovine serum to stop digestion; centrifuging for 5 minutes at 4 ℃ and 200g, discarding the supernatant to obtain a fifth precipitate, resuspending the fifth precipitate by using 10mL of cold Advanced DMEM/F-12, performing pipetting by using a 10mL pipette, pipetting organoids into smaller cell clusters, centrifuging for 5 minutes at 4 ℃ and 200g, discarding the supernatant to obtain a sixth precipitate, adding 200 μ L of precooled Advanced DMEM/F12 culture medium to resuspend the cell precipitates, adding 600 μ L of matrigel, inoculating, adding the culture medium for 8 days after the inoculated matrigel is solidified, and obtaining a passage thyroid cancer organoid, wherein the culture medium is replaced once every 3 days.
Second, minimum passable number statistics
The organoids obtained in example 3 were serially passaged as described above, and each time 30% of the organoids expanded to form a cell mass with a diameter exceeding 200 μm, the minimum passable number was recorded. The statistical results are shown in table 3.
Table 3: minimum passable times statistical table
| Culture medium | Minimum passable number of times |
| Formulation 1 | 8 |
| Formulation 2 | 8 |
| |
7 |
| Formulation 4 | 8 |
| Formulation 10 | 10 |
| Formulation 11 | 12 |
While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention within the context, spirit and scope of the invention. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.
Claims (10)
1. A culture medium for a thyroid cancer organoid, the culture medium comprising basal medium, B27, a p38MAPK inhibitor, and thyroid stimulating hormone; and the medium is free of at least one of Wnt3a, R-spondin-1, noggin, fibroblast growth factor, and A83-01.
2. The culture medium of claim 1, wherein the p38MAPK inhibitor comprises at least one selected from SB202190, SB203580, and Doramapimod; alternatively, the p38MAPK inhibitor is Doramapimod.
3. The culture medium of any one of claims 1-2, further comprising an epidermal growth factor.
4. The culture medium of any one of claims 1-3, wherein the concentration of the p38MAPK inhibitor in the culture medium is from 1 to 20 μ M, based on the total volume of the culture medium; and/or the volume percentage concentration of the B27 is 1-5%; and/or the concentration of said thyroid stimulating hormone is 1-50 ng/mL; and/or the concentration of the epidermal growth factor is 1-100 ng/mL.
5. The culture medium of any one of claims 1-4, further comprising at least one of N-acetylcysteine and nicotinamide.
6. The medium according to claim 5, wherein the concentration of N-acetylcysteine in the medium is 1-5mM based on the total volume of the medium; and/or the concentration of said nicotinamide is 1-20 mM.
7. Use of a culture medium according to any one of claims 1 to 6 for culturing a thyroid cancer organoid.
8. A culture method for culturing thyroid cancer organoids using the culture medium according to any one of claims 1 to 6, comprising the steps of:
(1) taking thyroid cancer tissues, cleaning, cutting into pieces, and digesting with digestive enzyme;
(2) terminating digestion; centrifuging, discarding the supernatant to obtain a first precipitate, re-suspending the first precipitate, filtering, centrifuging the filtrate, discarding the supernatant to obtain a second precipitate, re-suspending the second precipitate with an Advanced DMEM/F12 culture medium and matrigel, inoculating, and incubating;
(3) adding the culture medium to culture to obtain the thyroid cancer organoid.
9. The method of claim 8, the digestive enzymes comprising collagenase type II and/or pancreatin substitute.
10. The method according to any one of claims 8 to 9, wherein the ratio by volume of matrigel to Advanced DMEM/F12 medium is from 3:1 to 10: 1.
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