CN112831471A - Culture medium, culture method and detection method for thyroid cancer organoid - Google Patents

Culture medium, culture method and detection method for thyroid cancer organoid Download PDF

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CN112831471A
CN112831471A CN202110083883.7A CN202110083883A CN112831471A CN 112831471 A CN112831471 A CN 112831471A CN 202110083883 A CN202110083883 A CN 202110083883A CN 112831471 A CN112831471 A CN 112831471A
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culture medium
concentration
thyroid cancer
medium
culture
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黄卫人
陈栋
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Shenzhen Second Peoples Hospital
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Shenzhen Second Peoples Hospital
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Abstract

The invention provides a culture medium, a culture method and a detection method of thyroid cancer organoid, wherein the culture medium comprises a basic culture medium, B-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein and other components, and the culture medium has high culture success rate and high passage frequency on thyroid cancer organoid and has good passage stability after repeated cryopreservation and resuscitation; the thyroid cancer organoid obtained by adopting the culture medium and the culture method has high reducibility; the detection method is comprehensive and accurate.

Description

Culture medium, culture method and detection method for thyroid cancer organoid
Technical Field
The invention relates to the technical field of biomedicine, in particular to a culture medium, a culture method and a detection method for thyroid cancer organoids.
Background
Thyroid cancer is a common endocrine malignant tumor, and the incidence rate of the thyroid cancer is increased year by year in China, which belongs to high-incidence areas. Thyroid cancer can be classified into: papillary Thyroid Carcinoma (PTC), Follicular Thyroid Carcinoma (FTC), Medullary Thyroid Carcinoma (MTC), and Anaplastic Thyroid Carcinoma (ATC). Wherein PTC accounts for about 85% -90% of all thyroid cancer. 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.
At present, the individualized precise medical treatment of tumors lacks a proper preclinical evaluation model, and a 2D tumor cell line and a mouse transplantation model are not the optimal selection for the curative effect evaluation of preclinical antitumor drugs due to a plurality of defects. Organoids (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 progenitors, have stable phenotypic and genetic characteristics, and can be cultured in vitro for long periods of time. The tumor organoid model not only can show the biological characteristics of the tumor from which the model is derived, maintain the stability of gene expression, but also can accurately predict the response of a patient to an anti-tumor drug. At present, various tumor organoid culture systems of colorectal cancer, gastric cancer, prostate cancer, esophageal adenocarcinoma, pancreatic cancer, liver cancer, breast cancer, endometrial cancer, ovarian cancer, bladder cancer and the like are successfully established. The tumor organoid model from the patient is considered as a major breakthrough, and opens up a new visual field for the treatment of the individual cancer. At present, only 1 study on thyroid cancer organoids (Sondorp et al, cancers (Basel),2020,12(11):3212) is reported at home and abroad, but the thyroid cancer organoids are difficult to obtain in vitro long-term stable culture thyroid cancer models, and the culture steps, optimal culture medium formula and identification method of the thyroid cancer organoids are not specifically elucidated. The construction of the thyroid cancer organoid in-vitro model is of great significance for establishing a preclinical curative effect prediction platform of radioactive iodine and antitumor drugs, screening radioactive iodine and targeted drug treatment related genes, stopping ineffective iodine treatment in time, guiding patients to take drugs clinically and promoting the development of individualized and accurate medical treatment of refractory thyroid cancer.
Therefore, there is still a need to develop a culture medium, culture method and detection method for thyroid cancer organoids.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium, a culture method and a detection method of thyroid cancer organoids.
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 a basal medium and the following: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190 and thyroid stimulating hormone.
The culture medium may further contain at least one of fibroblast growth factor-7, fibroblast growth factor-10, Y-27632 and A83-01.
The basal medium can be Advanced DMEM/F12 medium.
In the culture medium, the percentage by volume of the B-27 may be 0.5-2% based on the total volume of the culture medium. In some embodiments, the percentage by volume of B-27 in the medium is 1% based on the total volume of the medium.
The concentration of the N-acetylcysteine in the culture medium may be 1-2mM based on the total volume of the culture medium. In some embodiments, the concentration of N-acetylcysteine in the medium is 1.25mM based on the total volume of the medium.
The concentration of nicotinamide in said culture medium may be between 1 and 50mM, based on the total volume of said culture medium. In some embodiments, the concentration of nicotinamide in said medium is 5-40mM, based on the total volume of said medium. In some embodiments, the concentration of nicotinamide in said culture medium is 5-20mM, based on the total volume of said culture medium. In some embodiments, the concentration of nicotinamide in said medium is 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 the culture medium, the concentration of the R-spondin 1 recombinant protein may be 1000ng/mL based on the total volume of the culture medium. In some embodiments, the concentration of the R-spondin 1 recombinant protein in the culture medium is 200-900ng/mL based on the total volume of the culture medium. In some embodiments, the concentration of the R-spondin 1 recombinant protein in the culture medium is 300-800ng/mL based on the total volume of the culture medium. In some embodiments, the concentration of the R-spondin 1 recombinant protein in the medium is 400-700ng/mL based on the total volume of the medium. In some embodiments, the concentration of the R-spondin 1 recombinant protein in the culture medium is 500-600ng/mL based on the total volume of the culture medium. In some embodiments, the concentration of the R-spondin 1 recombinant protein in the culture medium is 500ng/mL, based on the total volume of the culture medium.
The concentration of noggin in the medium may be 50-200ng/mL based on the total volume of the medium. In some embodiments, the concentration of the noggin in the medium is 150ng/mL, based on the total volume of the medium. In some embodiments, the concentration of noggin in the culture medium is 100ng/mL, based on the total volume of the culture medium.
The concentration of SB202190 in the culture medium may be 1-20 μ M based on the total volume of the culture medium. In some embodiments, the concentration of SB202190 in the culture medium is 5 to 15 μ M based on the total volume of the culture medium. In some embodiments, the concentration of SB202190 in the culture medium is 10 to 15 μ M, based on the total volume of the culture medium. In some embodiments, the concentration of SB202190 in the culture medium is 10 μ Μ, based on the total volume of the culture medium.
In the culture medium, the concentration of the A83-01 can be 1000nM, based on the total volume of the culture medium. In some embodiments, the concentration of A83-01 in the medium is 200 nM to 800nM, based on the total volume of the medium. In some embodiments, the concentration of A83-01 in the medium is 300-700nM, based on the total volume of the medium. In some embodiments, the concentration of A83-01 in the medium is 400-600nM, based on the total volume of the medium. In some embodiments, the concentration of a83-01 in the medium is 500nM, based on the total volume of the medium.
The concentration of the epidermal growth factor in the culture medium may be 10-100ng/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 fibroblast growth factor-7 may be present in the culture medium at a concentration of 1-10ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-7 is present in the culture medium at a concentration of 2-8ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-7 is present in the culture medium at a concentration of 3-7ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-7 is present in the culture medium at a concentration of 4-6ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-7 is present in the culture medium at a concentration of 5ng/mL, based on the total volume of the culture medium.
The fibroblast growth factor-10 may be present in the culture medium at a concentration of 1-10ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-10 is present in the culture medium at a concentration of 2-8ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-10 is present in the culture medium at a concentration of 3-7ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-10 is present in the culture medium at a concentration of 4-6ng/mL, based on the total volume of the culture medium. In some embodiments, the fibroblast growth factor-10 is present in the culture medium at a concentration of 5ng/mL, based on the total volume of the culture medium.
The concentration of the thyroid stimulating hormone in the medium may be 1 to 100. mu.g/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 10-90 μ g/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 20-80 μ g/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 30-70 μ g/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 40-60 μ g/mL based on the total volume of the medium. In some embodiments, the concentration of thyroid stimulating hormone in the medium is 50 μ g/mL based on the total volume of the medium.
In the culture medium, the concentration of Y-27632 may be 1-100. mu.M, based on the total volume of the culture medium. In some embodiments, the concentration of Y-27632 in the medium is 10-90 μ M based on the total volume of the medium. In some embodiments, the concentration of Y-27632 in the medium is 20-80 μ M based on the total volume of the medium. In some embodiments, the concentration of Y-27632 in the medium is 30-70 μ M based on the total volume of the medium. In some embodiments, the concentration of Y-27632 in the medium is 40-60 μ M based on the total volume of the medium. In some embodiments, the concentration of Y-27632 in the medium is 50 μ M based on the total volume of the medium.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid comprises a basal medium and the following: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190, and thyroid stimulating hormone; in the culture medium, the volume percentage of the B-27 is 0.5-2% based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1-2 mM; the concentration of the nicotinamide is 1-50 mM; the concentration of the R-spondin 1 recombinant protein is 100-1000 ng/mL; the concentration of the noggin is 50-200 ng/mL; the concentration of the epidermal growth factor is 10-100 ng/mL; the concentration of the SB202190 is 1-20 μ M; the concentration of the thyroid stimulating hormone is 1-100 mug/mL.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid comprises a basal medium and the following: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190, and thyroid stimulating hormone; the culture medium also contains at least one of fibroblast growth factor-7, fibroblast growth factor-10, Y-27632 and A83-01; in the culture medium, the volume percentage of the B-27 is 0.5-2% based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1-2 mM; the concentration of the nicotinamide is 1-50 mM; the concentration of the R-spondin 1 recombinant protein is 100-1000 ng/mL; the concentration of the noggin is 50-200 ng/mL; the concentration of the epidermal growth factor is 10-100 ng/mL; the concentration of the SB202190 is 1-20 μ M; the concentration of the thyroid stimulating hormone is 1-100 mug/mL; in the culture medium, the concentration of the fibroblast growth factor-7 is 1-10ng/mL based on the total volume of the culture medium; the concentration of the fibroblast growth factor-10 is 1-10 ng/mL; the concentration of the Y-27632 is 1-100 mu M; the concentration of A83-01 is 100-1000 nM.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid comprises a basal medium and the following: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190, and thyroid stimulating hormone; in the culture medium, the volume percentage of the B-27 is 1 percent based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1.25 mM; the concentration of nicotinamide is 10 mM; the concentration of the R-spondin 1 recombinant protein is 500 ng/mL; the concentration of the noggin is 100 ng/mL; the concentration of the epidermal growth factor is 50 ng/mL; the concentration of the SB202190 is 10 μ M; the concentration of the thyroid stimulating hormone is 10 mug/mL.
In some embodiments of the invention, a culture medium for a thyroid cancer organoid comprises a basal medium and the following: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190, and thyroid stimulating hormone; the culture medium also contains at least one of fibroblast growth factor-7, fibroblast growth factor-10, Y-27632 and A83-01; in the culture medium, the volume percentage of the B-27 is 1 percent based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1.25 mM; the concentration of nicotinamide is 10 mM; the concentration of the R-spondin 1 recombinant protein is 500 ng/mL; the concentration of the noggin is 100 ng/mL; the concentration of the epidermal growth factor is 50 ng/mL; the concentration of the SB202190 is 10 μ M; the concentration of the thyroid stimulating hormone is 10 mug/mL; in the culture medium, the concentration of the fibroblast growth factor-7 is 5ng/mL based on the total volume of the culture medium; the concentration of the fibroblast growth factor-10 is 5 ng/mL; the concentration of the Y-27632 is 10 mu M; the concentration of A83-01 was 10 nM.
In a second aspect, the present invention provides a method for culturing thyroid cancer organoids.
A method for culturing thyroid cancer organoids by using the culture medium comprises the following steps:
(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 a 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 may include collagenase type II and/or pancreatin substitutes.
The volume ratio of the matrigel to the DMEM/F12 culture medium can be 3:1-10: 1.
The culture may be carried out at 37 ℃ for 6-14 days, with the medium being changed every 3-5 days.
In some embodiments of the invention, a method of culturing thyroid cancer organoids in the aforementioned culture medium 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 DMEM/F12 culture medium to mix with the second precipitate, adding matrigel to re-suspend, wherein the volume ratio of the matrigel to the DMEM/F12 culture medium is 3:1-10:1, inoculating, and incubating;
(3) adding the culture medium, culturing at 37 ℃ for 7 days, and replacing the culture medium every 3 days to obtain the thyroid cancer organoid.
In a third aspect, the present invention provides a method for detecting a thyroid cancer organoid.
A method for detecting thyroid cancer organoids obtained by the above method, comprising the steps of: and taking the obtained thyroid cancer organoid and a thyroid cancer tissue for preparing the thyroid cancer organoid, and respectively carrying out hematoxylin-eosin staining, immunohistochemical staining, whole genome sequencing and exome sequencing.
The identification marker of immunohistochemical staining may comprise a marker selected from the group consisting of: at least one of galectin 3, cytokeratin 19, HBME-1, thyroglobulin, calcitonin, thyroid transcription factor 1, matrix metalloproteinase, Ki-67, CD117, and p 53.
The data size for the whole exon sequencing may be 24G.
The depth of the whole exon sequencing may be 200X.
The similarity of the thyroid cancer organoid and the thyroid cancer tissue for preparing the thyroid cancer organoid in gene mutation and copy number variation is more than 80%.
Advantageous effects
Compared with the prior art, the invention has the following beneficial effects:
1. the culture medium, the culture method and the identification method of the thyroid cancer organoid are the optimal culture medium, the operation process and the identification method which are designed aiming at the culture characteristics of thyroid cancer cells and the pathophysiological characteristics of the thyroid cancer organoid.
2. 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 organoids in a 3D environment, the culture success rate can reach 92%, and the number of passages can reach at least 15.
3. The thyrotropin is added into the culture medium, so that the culture success rate and the passage times of thyroid cancer organoids are improved.
4. The culture system established by the invention can stably culture thyroid cancer organoids in vitro for a long time, and still maintain high growth efficiency after repeated cryopreservation and resuscitation.
5. 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.
6. The invention simultaneously carries out multi-dimensional detection on thyroid cancer organoids on histopathology and genomics, shows that the thyroid cancer organoids can highly reduce the characteristics of parent tumors of patients, and shows that the thyroid cancer organoids can be effectively used as in-vitro 'substitute' of in-vivo tumors for further research.
7. 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.
Drawings
FIG. 1 is a brightfield image and hematoxylin-eosin (H & E) staining of thyroid cancer organoids cultured in the medium described in example 3 using the medium described in example 1; the scale bar is 100 μm.
FIG. 2 is a hematoxylin-eosin (H & E) staining picture and a bright field photograph of thyroid cancer organoids and tumor tissues derived therefrom, which were cultured in the medium described in example 3 according to example 1; the scale bar is 100 μm.
FIG. 3 is an image of immunohistochemical staining of thyroid cancer organoids and tumor tissues derived therefrom cultured in the medium described in example 3 in example 1; the scale bar is 100 μm.
FIG. 4 is a genomics analysis of thyroid cancer organoids and their tumor of origin cultured in example 3 using the medium described in example 1; a represents a comparison hotspot graph of high-frequency mutant genes of thyroid cancer tissues and corresponding organoids; b represents a histogram comparing the type of point mutations of thyroid cancer tissues and corresponding organoids; c, comparing the gene copy number variation of thyroid cancer tissues and corresponding organoids; in which patients 2, 4 and 6 were sampled from different locations of thyroid cancer tissue to establish two thyroid cancer organoid lines, respectively.
FIG. 5 is a hematoxylin-eosin (H & E) staining picture and a bright field photograph of thyroid cancer organoids and tumor tissues derived therefrom cultured in the medium of example 2 in example 3; the scale bar is 100 μm.
FIG. 6 is a photograph of immunohistochemical staining of thyroid cancer organoids and tumor tissues derived therefrom cultured in the medium described in example 3 in example 2; the scale bar is 100 μm.
FIG. 7 is a flow chart showing the culture and detection of thyroid cancer organoids in examples 3 and 4.
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, CK19 represents cytokeratin 19; galectin-3 represents Galectin 3; tg means thyroglobulin; TTF-1 represents thyroid transcription factor 1; CD117 represents a tyrosine kinase receptor protein; ki-67 is a nuclear protein associated with cell proliferation.
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:
Figure BDA0002910246720000081
Figure BDA0002910246720000091
Figure BDA0002910246720000101
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 medium
A culture medium for thyroid cancer organoids, the culture medium consisting of Advanced DMEM/F12 medium and the following components: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190, A83-01, fibroblast growth factor-7, fibroblast growth factor-10, thyroid stimulating hormone, and Y-27632; in the culture medium, the volume percentage of the B-27 is 1 percent based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1.25 mM; the concentration of nicotinamide is 10 mM; the concentration of the R-spondin 1 recombinant protein is 500 ng/mL; the concentration of the noggin is 100 ng/mL; the concentration of the epidermal growth factor is 50 ng/mL; the concentration of the SB202190 is 10 μ M; the concentration of the A83-01 is 500 nM; the concentration of the fibroblast growth factor-7 is 5 ng/mL; the concentration of the fibroblast growth factor-10 is 5 ng/mL; the concentration of the thyroid stimulating hormone is 10 mug/mL; the concentration of the Y-27632 is 10 mu M, and the balance is Advanced DMEM/F12 culture medium.
Example 2: culture medium
A culture medium for thyroid cancer organoids, the culture medium consisting of Advanced DMEM/F12 medium and the following components: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190, and thyroid stimulating hormone; in the culture medium, the volume percentage of the B-27 is 1 percent based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1.25 mM; the concentration of nicotinamide is 10 mM; the concentration of the R-spondin 1 recombinant protein is 500 ng/mL; the concentration of the noggin is 100 ng/mL; the concentration of the epidermal growth factor is 50 ng/mL; the concentration of the SB202190 is 10 μ M; the concentration of the thyroid stimulating hormone is 10 mug/mL; the balance was Advanced DMEM/F12 medium.
Example 3: culture of thyroid cancer organoids
The operation is as follows: the culture medium of example 1 or 2 is taken respectively, and the thyroid cancer tissues of 8 different patients obtained clinically are taken respectively, and the thyroid cancer organoids are cultured according to the following steps:
(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 with Hanks liquid, filtering with a 70-micron cell filter screen, counting cells, centrifuging filtrate, discarding supernatant to obtain a second precipitate, mixing with a DMEM/F12 culture medium and the second precipitate, adding matrigel for re-suspending, wherein the volume ratio of the matrigel to the DMEM/F12 culture medium is 3:1, inoculating, incubating for 2 minutes, and inverting for 8 minutes to solidify the matrigel;
(3) adding the culture medium, culturing at 37 ℃ for 8 days, and replacing the culture medium every 3 days to obtain the thyroid cancer organoid.
As a result: thyroid cancer organoids obtained using the medium described in example 1 are shown in FIGS. 1 and 2; the thyroid cancer organoids obtained using the medium described in example 2 are shown in FIG. 5.
And (4) analyzing results:
(1) FIG. 1 shows that thyroid cancer organoids can reach a diameter of 100-150 μm after 14 days of culture using the medium described in example 1, and further expand to 200 μm or more after 21 days of culture; in addition, thyroid cancer organoids are mostly found to grow as single cells in a single clone.
(2) FIGS. 2 and 5 are middle columns showing bright field pictures of thyroid cancer organoids successfully cultured using the media described in examples 1 and 2, respectively; under the observation of light, thyroid cancer organoids are mostly in the form of compact solid spheres, and have irregular shapes as shown in patients 2 and 4 in fig. 2.
Example 4: detection of thyroid cancer organoids
The operation is as follows: taking the thyroid cancer organoids obtained in the embodiment 3 and thyroid cancer tissues from the thyroid cancer organoids, performing hematoxylin-eosin staining and immunohistochemical staining, and analyzing whether the organoids reproduce the characteristics of tumor tissues from the thyroid cancer organoids in aspects of morphology, histopathology, tumor marker expression and the like; taking the cultured thyroid cancer organoid and the thyroid cancer tissue from the thyroid cancer organoid, extracting genome DNA, performing whole genome exon library construction and sequencing, and checking whether the cultured thyroid cancer organoid can reduce the characteristics of gene mutation, copy number variation and the like of the tumor from which the thyroid cancer organoid is derived. Wherein, the differentiation markers of thyroid cancer tissues and organoids comprise: galectin 3(Galectin-3), cytokeratin 19(CK19), thyroglobulin (Tg), thyroid transcription factor 1(TTF-1), Ki-67 and CD 117; the data volume for sequencing all exons of thyroid cancer tissues and organoids was 24G with a depth of 200X.
As a result: as shown in fig. 2-6.
And (4) analyzing results:
(1) FIGS. 2 and 5 show bright field images of thyroid cancer organoids cultured in the media described in examples 1 and 2, respectively, and a hematoxylin-eosin staining contrast to the tumor tissue from which it was derived, indicating that thyroid cancer organoids are very similar in morphology and staining characteristics to their tumor tissue from which it was derived.
(2) FIGS. 3 and 6 show immunohistochemical profiles of thyroid cancer organoids and their tumor tissues of origin, in culture with the medium described in examples 1 and 2, respectively, indicating the pathology and marker protein expression characteristics of thyroid cancer organoids capable of reducing the tumor tissues of origin.
(3) FIG. 4 shows the characteristics of thyroid cancer organoids that are capable of highly reducing their tumor of origin in terms of gene mutation and copy number variation.
The results show that the thyroid cancer organoid established by the invention can highly reduce the histopathology and genomics characteristics of parent tumor, and the detection method is comprehensive and accurate.
Comparative example 1: culture medium (refer to CN 111411083B medium formula)
A culture medium, comprising a basic culture medium 1640, specific additive factors and sterile water; wherein the mass ratio of the basic culture medium 1640 to the sterile water is 99: 1; the specific addition factors comprise: vitamin a-free B27, 2X; n-acetyl cysteine, 0.5. mu.M; EGF, 50 ng/mL; noggin, 100 ng/mL; r-spondin 1, 800 ng/mL; wnt3a, 100 ng/mL; CHIR99021, 8 μ M; 1.5 μ M of thiazovivin; gastrin I, 25 ng/mL; valproic acid, 0.5 mM; penicillin streptomycin mixed solution, 1.2X; amphotericin B, 0.8. mu.g/mL; primocin, 1 mg/mL; the final concentration of each component of the specific additive factor is based on the final concentration of each component in the mixed solution of the basic culture medium and the sterile water.
Comparative example 2: culture medium (refer to CN109837242A medium formula)
A culture medium comprising 1% penicillin, 1% streptomycin, 50ng/mL EGF, 50ng/mL fibroblast growth factor, 50ng/mL Noggin recombinant protein, 20mM HEPES, 500nM A83-01, 1.0 μ g/mL R-spondin 3, 5 μ M Y-27632, 5 μ M SB202190, 1% B27, 3mM Glutamax, Advanced DMEM/F12 medium with 8% FBS by volume.
Comparative example 3: culture medium (refer to CN108396010A medium formula)
A culture medium comprising 50ng/mL EGF, 100ng/mL Wnt-3a, 1. mu.g/mL R-spondin 1, 500nM A83-01, 10. mu.M Y-27632, 50ng/mL Noggin recombinant protein, 12. mu.M SB202190, 1X N2, 1X B27 supplement, 10mM HEPES, 2mM Glutamax, 500 units/mL penicillin, 500 units/mL streptomycin, 12.5. mu.g/mL amphotericin, DMEM/F12 medium containing 10% by volume of FBS.
Comparative example 4: culture medium (refer to CN111876386A medium formula)
The media were prepared as in table 1:
table 1: formula of culture medium
Reagent Final concentration
R-spondin 1 conditioned medium 250ng/mL
Neuregμlin
1 5nM
EGF 5ng/mL
Noggin recombinant protein 100ng/mL
A83-01 500nM
Y-27632 5μM
SB202190 500nM
B27supplement(50X) 1X
N-acetylcysteine 1.25mM
Nicotinamide 5mM
GlutaMAX(100X) 1X
HEPES(100X) 1X
Penicillin/streptomycin 1X
Primocin 50mg/mL
Advanced DMEM/F-12 1X
Comparative example 5: thyrotropin-deficient medium
A culture medium for thyroid cancer organoids, the culture medium consisting of Advanced DMEM/F12 medium and the following components: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190, A83-01, fibroblast growth factor-7, fibroblast growth factor-10, thyroid stimulating hormone, and Y-27632; in the culture medium, the volume percentage of the B-27 is 1 percent based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1.25 mM; the concentration of nicotinamide is 10 mM; the concentration of the R-spondin 1 recombinant protein is 500 ng/mL; the concentration of the noggin is 100 ng/mL; the concentration of the epidermal growth factor is 50 ng/mL; the concentration of the SB202190 is 10 μ M; the concentration of the A83-01 is 500 nM; the concentration of the fibroblast growth factor-7 is 5 ng/mL; the concentration of the fibroblast growth factor-10 is 5 ng/mL; the concentration of the Y-27632 is 10 mu M, and the balance is Advanced DMEM/F12 culture medium.
Comparative example 6: organoid culture
The operation is as follows: the culture media of comparative examples 1-5 were used to culture organoids according to the following procedure:
(1) collecting clinically obtained thyroid cancer tissue, cleaning, 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 with Hanks liquid, filtering with a 70-micron cell filter screen, counting cells, centrifuging filtrate, discarding supernatant to obtain a second precipitate, adding a DMEM/F12 culture medium to mix with the second precipitate, adding matrigel to carry out re-suspension, wherein the volume ratio of the matrigel to the DMEM/F12 culture medium is 3:1, inoculating, incubating for 2 minutes, and then inverting for 8 minutes to solidify the matrigel;
(3) adding the culture medium, culturing at 37 ℃ for 8 days, and replacing the culture medium every 3 days to obtain the thyroid cancer organoid.
Example 5: culture success rate of different culture media
Thyroid cancer tissues of different patients were collected, and the culture media of example 1, example 2, and comparative example 1 to comparative example 5 were cultured in the culture methods of example 3 and comparative example 6 for 25 cases, respectively. The culture success rates of the respective media and the culture methods thereof are shown in table 2.
Table 2: culture success rate of different culture media
Figure BDA0002910246720000141
Figure BDA0002910246720000151
Example 6: 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 example 1, example 2, comparative example 1 to comparative example 5 (the medium used for passaging is the same as the medium used when organoids are cultured).
The operation is as follows:
centrifuging the thyroid cancer organoids obtained in example 3 or comparative example 6 at 4 deg.C and 200g for 5 min, discarding the supernatant to obtain a fourth precipitate, adding the second digestion solution to the fourth precipitate, and digesting with a shaker at 37 deg.C and 120rpm for 5 min; adding Advanced DMEM/F12 medium containing 20% by volume fetal bovine serum to stop digestion; centrifuging for 5 minutes at 4 ℃ under the condition of 200g, discarding the supernatant to obtain a fifth precipitate, resuspending the fifth precipitate by using 10mL of cold Advanced DMEM/F-12, blowing by using a 10mL pipette to blow the organoid into a smaller cell mass, centrifuging for 5 minutes at 4 ℃ under the condition of 200g, discarding the supernatant to obtain a sixth precipitate, adding 200 mu L of precooled DMEM/F12 culture medium to resuspend the cell precipitate, adding 600 mu L of matrigel, inoculating, adding the culture medium for 8 days after the inoculated matrigel is solidified to obtain the subculture thyroid cancer organoid, and replacing the culture medium once every 3 days.
Second, minimum passable number statistics
The organoids obtained in example 3 and comparative example 6 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
Example 1 15 times of
Example 2 14 times (twice)
Comparative example 1 6 times of
Comparative example 2 7 times (twice)
Comparative example 3 6 times of
Comparative example 4 8 times (by volume)
Comparative example 5 9 times of
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 a basal medium and the following: b-27, N-acetylcysteine, nicotinamide, R-spondin 1 recombinant protein, noggin, epidermal growth factor, SB202190 and thyroid stimulating hormone.
2. The culture medium of claim 1, further comprising at least one of fibroblast growth factor-7, fibroblast growth factor-10, Y-27632, and a 83-01.
3. The culture medium according to any one of claims 1-2, wherein the percentage by volume of B-27 in the culture medium is 0.5-2% based on the total volume of the culture medium; the concentration of the N-acetylcysteine is 1-2 mM; the concentration of the nicotinamide is 1-50 mM; the concentration of the R-spondin 1 recombinant protein is 100-1000 ng/mL; the concentration of the noggin is 50-200 ng/mL; the concentration of the epidermal growth factor is 10-100 ng/mL; the concentration of the SB202190 is 1-20 μ M; the concentration of the thyroid stimulating hormone is 1-100 mug/mL.
4. The culture medium according to any one of claims 2 to 3, wherein the fibroblast growth factor-7 is present in the culture medium at a concentration of 1 to 10ng/mL, based on the total volume of the culture medium; the concentration of the fibroblast growth factor-10 is 1-10 ng/mL; the concentration of the Y-27632 is 1-100 mu M; the concentration of A83-01 is 100-1000 nM.
5. A method for culturing thyroid cancer organoids in the medium according to any one of claims 1 to 4, 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 a DMEM/F12 culture medium and matrigel, inoculating, and incubating;
(3) adding the culture medium to culture to obtain the thyroid cancer organoid.
6. The method of claim 5, the digestive enzymes comprising collagenase type II and/or pancreatin substitute.
7. The method according to any one of claims 5 to 6, wherein the volume ratio of the matrigel to the DMEM/F12 medium is 3:1 to 10: 1.
8. A method for detecting thyroid cancer organoids according to any one of claims 5 to 7, comprising the steps of: and taking the obtained thyroid cancer organoid and a thyroid cancer tissue for preparing the thyroid cancer organoid, and respectively carrying out hematoxylin-eosin staining, immunohistochemical staining, whole genome sequencing and exome sequencing.
9. The detection method of claim 8, the identification marker of immunohistochemical staining comprising a marker selected from the group consisting of: at least one of galectin 3, cytokeratin 19, HBME-1, thyroglobulin, calcitonin, thyroid transcription factor 1, matrix metalloproteinase, Ki-67, CD117, and p 53.
10. The detection method according to any one of claims 8 to 9, wherein the data size of the whole exon sequencing is 24G, and the depth of the whole exon sequencing is 200 x.
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