CN118345044A - Cervical squamous carcinoma organoid culture medium and culture method - Google Patents

Abstract

The invention belongs to the technical field of organoid culture, and particularly relates to a cervical squamous carcinoma organoid culture medium and a culture method. The cervical squamous carcinoma organoid medium comprises the following components: basal medium, HEPES buffer, nutritional additives and specific active factors; the specific active factors comprise penicillin-streptomycin, amphotericin B, noggin, nicotinamide, N-acetylcysteine, ROCK inhibitor, A83-01, FGF7, SB202190, EGF, hydrocortisone, insulin transferrin selenium ITS, jagged-1, PGE2 and MHY1485. When the cervical squamous carcinoma organoid culture medium is combined with the improved organoid culture method for culturing the cervical squamous carcinoma organoid, a larger number of tumor organoids can be obtained, and the success rate and the formation efficiency of the cervical squamous carcinoma organoid culture are obviously improved.

Description

Cervical squamous carcinoma organoid culture medium and culture method

Technical Field

The invention belongs to the technical field of organoid culture, and particularly relates to a cervical squamous carcinoma organoid culture medium and a culture method.

Background

Cervical cancer is the most common gynaecological malignancy, and is an important cause of cancer death in women. In 2020, about 60.4 ten thousand women worldwide were diagnosed with cervical cancer and 34.2 ten thousand died from the disease. Squamous cell carcinoma is the major pathological type, accounting for over 70%. At present, prevention measures such as HPV vaccine inoculation, cervical cytology smear examination and HPV-DNA detection are carried out regularly worldwide, so that cervical cancer can be discovered and treated early. However, cervical cancer incidence is still high in low income areas. Cervical cancer research depends on a limited number of cell lines (such as HeLa or CaSki), xenograft animal models and the like, which have limited value in clinical research after multiple passages and species differences, and cannot simulate the true pathophysiological state of tumors in vitro. This results in the failure of the efficacy of the basic experimental study to be reproduced in clinical treatment.

Organoids are 3D clusters of cells grown from (pluripotent or adult) stem cells and spontaneously organize into organ-like or tissue-like structures that are enriched for the cell types typically found in the tissue of origin. Compared with the traditional two-dimensional culture system, the three-dimensional organoid system is closer to the original tissue or organ in terms of gene and protein expression, metabolic function, microstructure and the like. In tumor research, organoids not only reflect the structural and functional characteristics of the source tumor tissue, but also remain highly similar to the source tissue at the genomic level, reproducing tumor characteristics such as copy number variation, recurrent mutations, tumor heterogeneity, and the like. Genetic stability can be maintained even after long passage. Therefore, the organoids are used as miniaturized and simplified organ model systems, have quite successful in disease modeling, accurate medical treatment and drug screening, and provide a valuable pathological model for tumor research.

Developmental biology studies have revealed that cytokines determine cell fate at different stages during embryonic development. The behavior of cells in vivo depends on microenvironment signals and contact with neighboring cells and extracellular matrix (ECM). Organoid technology relies on a mixture of various growth factors and small molecules to reconstruct the niche for long-term survival of (pluripotent or adult) stem cells. Under regulation of signaling pathways directed against key regulatory effects, cells undergo proliferation, differentiation, migration and selection. This also allows organoids to further surpass two-dimensional cell cultures in terms of structural similarity and functional reproduction with the corresponding organ. Therefore, it is important to determine the composition of the organoid medium to maintain the structural and functional characteristics of the source organ. The maintenance of stem cell niches by the organoid culture fluids currently in common use is based on Wnt, rspondin, EGF, which facilitates the culture of most normal tissue organoids, such as human and mouse small intestine, stomach, liver, pancreas, etc. However, isolated culture of human tumor tissue organoids still faces the problems of low organoid survival and high failure rate. In addition, the separation method of filtering and collecting single cell suspension after digestion is adopted, so that the utilization rate of the sample is low, because more undigested tumor tissues can be discarded, the digestion time is prolonged, and the cell activity is damaged. Therefore, it is necessary to develop a new cervical squamous carcinoma organoid culture medium and a new culture method, so as to improve the success rate and the formation efficiency of organoid isolated culture, and provide a premise for the wide application of organoid technology.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings of the prior art and provide a cervical squamous carcinoma organoid culture medium and a culture method thereof, so as to improve the success rate and the formation efficiency of organoid separation culture.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

In a first aspect, the invention features a cervical squamous carcinoma organoid medium comprising the following components: basal medium, HEPES buffer, nutritional additives and specific active factors; the specific active factors comprise penicillin-streptomycin, amphotericin B, noggin, nicotinamide, N-acetylcysteine, ROCK inhibitor, A83-01, FGF7, SB202190, EGF, hydrocortisone, insulin transferrin selenium ITS, jagged-1, PGE2 and MHY1485; the nutritional additives include Glutamax, N2 and B27.

N2 adopted in the culture medium can be used as a substitute factor of fetal bovine serum, and the chemical components are definite; ITS facilitates transport of nutrients and antioxidant; jagged-1 can activate Notch pathway and promote malignant biological behavior of tumor cells; PGE2 can enhance the stem properties of tumor cells; MHY1485 is able to activate mTOR signaling pathway, promoting metabolic activity and proliferation of cervical squamous carcinoma cells. Therefore, the invention is beneficial to maintaining the ecological niche of cervical squamous carcinoma cells and effectively promoting the formation and growth of cervical squamous carcinoma organoids by removing the inhibition factors of cervical squamous carcinoma organoids and adding various signal molecules for promoting the occurrence and progress of cervical carcinoma as the cervical squamous carcinoma cell culture medium.

Preferably, the cervical squamous carcinoma organoid medium comprises the following components: basal medium, 8-12mM HEPES buffer, 0.5-1.5 XGlutamax, 0.5-1% N2, 0.5-1.5 XB 27, 0.5-1.5% penicillin-streptomycin, 0.5-1.5% amphotericin B, 0.5-1% Noggin, 1-2.5mM nicotinamide, 0.5-1.25mM N-acetylcysteine, 8-12. Mu.M ROCK inhibitor, 200-500nM A83-01, 10-25ng/ml FGF7, 0.1-1. Mu.M SB202190, 200-500. Mu.g/ml EGF, 100-250. Mu.g/ml hydrocortisone, 0.5-1% insulin transferrin selenium ITS, 0.1-1. Mu.M Jagged-1, 0.1-1. Mu.M PGE2 and 0.5-2. Mu.M MHY 5.

More preferably, the cervical squamous carcinoma organoid medium comprises the following components: basal medium, 10mM HEPES buffer, 1 XGlutamax, 1% N2, 1 XB 27, 1% penicillin-streptomycin, 1% amphotericin B, 1% Noggin, 2.5mM nicotinamide, 1.25mM N-acetylcysteine, 10. Mu.M ROCK inhibitor, 500nM A83-01, 25ng/ml FGF7, 1. Mu.M SB202190, 500. Mu.g/ml EGF, 250. Mu.g/ml hydrocortisone, 1% insulin transferrin selenium ITS,. Mu.M Jagged-1, 1. Mu.M PGE2 and 2. Mu.M MHY1485.

The inventor finds that the success rate and the efficiency of the cervical squamous carcinoma organoid culture can be improved more favorably when the concentration of each component in the cervical squamous carcinoma cell culture medium adopts the more preferable value.

Preferably, the basal medium is DMEM or DMEM/F12.

Preferably, the cervical squamous carcinoma organoid medium further comprises the following components: 0.5-1.5% L-glutamic acid, 0.5-1.5% L-alanine or 0.5-1.5% glycine.

In a second aspect, the present invention provides a method for culturing cervical squamous carcinoma organoids, comprising the steps of:

(1) Collecting cervical squamous cell carcinoma tissue, cleaning to remove blood clots and mucus, and shearing the carcinoma tissue into fine particles;

(2) Adding mixed solution containing IV collagenase and EDTA into sheared cancer tissue, shaking table digestion for 10-15min, standing for 1-3min, collecting supernatant, and stopping digestion with serum-free culture solution;

(3) Repeating the digestion and standing process of the step (2) on the rest cancer tissue sediment, collecting the supernatant and stopping the digestion until the cancer tissue appears as mucus flocculent;

(4) Centrifuging the repeatedly collected supernatant to obtain cell precipitate, adding Matrigel for resuspension, inoculating, fixing, adding the above cervical squamous carcinoma organoid culture medium, and culturing to obtain the cervical squamous carcinoma organoid.

The method for culturing the cervical squamous carcinoma organoid adopts a method of repeated standing after digestion to collect cervical squamous carcinoma tumor cells, can obtain more dispersed single tumor cells and tumor cell clusters, and greatly improves the utilization rate of specimens, so that a larger number of tumor organoids can be obtained after the culture of the cervical squamous carcinoma organoid culture medium, and the success rate and the formation efficiency of the cervical squamous carcinoma organoid culture are obviously improved.

Preferably, in the step (2), the concentration of collagenase type IV is 1mg/ml and the concentration of EDTA is 30nM.

Through experimental research, the efficiency is higher when the mixed solution of the IV collagenase and the EDTA in the preferable concentration range is adopted for digestion and collection of cervical squamous cell carcinoma tumor cells.

Preferably, in the step (4), the volume ratio of the cell pellet to Matrigel is 1:2-1:3.

More preferably, in the step (4), the volume ratio of the cell pellet to Matrigel matrix gel is 1:2.

In a third aspect, the invention provides the use of the above-described culture medium for culturing cervical squamous carcinoma organoids.

Compared with the prior art, the invention has the following beneficial effects:

According to the invention, by adding various specific signal molecules for promoting the occurrence and the progress of cervical cancer into the cervical squamous carcinoma organoid culture medium, the ecological niche of cervical squamous carcinoma cells can be maintained, and the formation and the growth of cervical squamous carcinoma organoids can be promoted; in addition, the invention uses IV collagenase and EDTA mixed solution for digestion, and adopts a culture method of repeated standing after digestion to collect cervical squamous carcinoma tumor cells, so that more dispersed single tumor cells and tumor cell clusters can be obtained, and the utilization rate of the specimen is greatly improved; when the improved organoid culture method is combined with the cervical squamous carcinoma organoid culture medium to culture the cervical squamous carcinoma organoids, a larger number of tumor organoids can be obtained, and the success rate and the formation efficiency of the cervical squamous carcinoma organoids can be obviously improved.

Drawings

FIG. 1 is a schematic diagram of isolated culture of cervical squamous carcinoma organoids, wherein A is the cyclic digestion and isolation process of cervical squamous cell carcinoma tissue, and B is the culture broth culture of cervical squamous carcinoma organoids with addition of various specific active factors.

Fig. 2 is a schematic diagram showing the cultivation and identification of cervical squamous carcinoma organoids, wherein fig. a is a bright field image of cervical squamous carcinoma organoids cultivated in example 1+example 6 and example 2+example 6; panel B is a HE staining and immunohistochemical staining pattern of cervical squamous carcinoma organoids; c is an electron microscope image of cervical squamous carcinoma organoid after subculture, with a scale of 100 μm; and D is an electron microscope image after cryopreservation and resuscitation of cervical squamous carcinoma organoids, and the scale is 100 mu m.

FIG. 3 is a graph of the comparative cervical squamous carcinoma organoids of examples and comparative examples, wherein graph A is a plot of the dead/alive staining of the cervical squamous carcinoma organoids, to a scale of 50 μm; panel B is a comparison of the number of isolated cultured cervical squamous carcinoma organoids of example 6 and comparative example 3; panel C is a bright field image of a cervical squamous carcinoma organoid cultured in example 1 and comparative example 1, scale bar 100 μm; panel D is a graph comparing the organogenesis efficiency of cervical squamous carcinoma of example 1 and comparative example 1; FIG. E is a graph showing the percentage of cervical squamous carcinoma organoids having a diameter of 200 μm or more in example 1 and comparative example 1; FIG. F is a graph showing the effect of different specific active factors on the organogenesis efficiency of cervical squamous carcinoma; g is a graph comparing the proliferative activity of cervical squamous carcinoma organoids constructed in example 1+example 6 and comparative example 1+comparative example 3.

Detailed Description

The above-described aspects of the present invention will be described in further detail by way of specific embodiments of the present invention. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples.

The reagents used in the examples, unless otherwise specified, are all conventional reagents known in the art and are commercially available. The experimental operations not specifically described in the examples are all conventional operations in the art or are understood or known by those skilled in the art from the prior art or common general knowledge in which they are well known.

MEM/F12 in the cervical squamous carcinoma organoid culture medium is purchased from Gibco ^TM, inc., 320033; glutamax is purchased from Gibco,35050061; n2 was purchased from Invitrogen,75510-019-5; b27 is purchased from Gibco corporation, 175044; penicillin-streptomycin is purchased from Gibco corporation, 10378016; amphotericin B was purchased from Gibco corporation, 15290026; noggin is purchased from U-Protein Express, N002; nicotinamide was purchased from Sigma, N0636; n-acetylcysteine was purchased from Sigma company; a9165; ROCK inhibitors were purchased from Stemcell Inc., 129830-38-2; a83-01 is purchased from Tocres, 2939; FGF7 is purchased from Peprotech,100-19; SB202190 was purchased from Sigma,7067; EGF was purchased from Sigma, SRP3027; hydrocortisone is purchased at aladdin, H110523; insulin transferrin selenium ITS is purchased from Gibco,51500056; jagged-1 was purchased from Sigma, SRP8012; PGE2 was purchased from Sigma,900117P and MHY1485 from Sigma, SML0810.

Example 1

The embodiment provides a cervical squamous carcinoma organoid culture medium, which comprises the following components:

DMEM/F12, 10mM HEPES buffer, 1 XGlutamax, 1% N2, 1 XB 27, 1% penicillin-streptomycin, 1% amphotericin B, 1% Noggin, 2.5mM nicotinamide, 1.25mM N-acetylcysteine, 10. Mu.M ROCK inhibitor, 500nM A83-01, 25ng/ml FGF7, 1. Mu.M SB202190, 500. Mu.g/ml EGF, 250. Mu.g/ml hydrocortisone, 1% insulin transferrin selenium ITS, 1. Mu.M Jagged-1, 1. Mu.MPGE 2 and 2. Mu.M MHY1485.

The preparation method of the cervical squamous carcinoma organoid culture medium comprises the following steps:

Firstly adding DMEM/F12 basal medium into a 50ml centrifuge tube, adding 1 XB 27, 1% N2, 1xGlutamax, 10mM HEPES buffer solution, 2.5mM nicotinamide and 1.25mM N-acetylcysteine, uniformly mixing, then adding other growth factors, and sequentially adding 1 mu M SB202190, 1% Noggin, 500nM A83-01, 25ng/ml FGF7, 500 mu g/ml EGF, 250 mu g/ml hydrocortisone, 1% insulin transferrin selenium ITS, 1 mu M Jagged-1, 1 mu M PEG2, 2 mu M MHY1485 and 10 mu M ROCK inhibitor, and finally adding 1% penicillin-streptomycin and 1% amphotericin B to obtain the cervical squamous carcinoma organoid culture medium.

Example 2

The embodiment provides a cervical squamous carcinoma organoid culture medium, which comprises the following components:

DMEM/F12, 10mM HEPES buffer, 1 XGlutamax, 0.5% N2, 1 XB 27, 1% penicillin-streptomycin, 1% amphotericin B, 0.5% Noggin, 1mM nicotinamide, 0.5mM N-acetylcysteine, 10. Mu.M ROCK inhibitor, 200nM A83-01, 10ng/ml FGF7, 0.1. Mu.M SB202190, 200. Mu.g/ml EGF, 100. Mu.g/ml hydrocortisone, 0.5% insulin transferrin selenium ITS, 0.1. Mu.M Jagged-1, 0.1. Mu.MPGE 2 and 0.5. Mu.M MHY1485.

The preparation method of the cervical squamous carcinoma organoid culture medium adopts the preparation method described in the embodiment 1.

Example 3

The embodiment provides a cervical squamous carcinoma organoid culture medium, which comprises the following components:

DMEM/F12, 8mM HEPES buffer, 0.5 XGlutamax, 0.5% N2, 0.5 XB 27, 0.5% penicillin-streptomycin, 0.5% amphotericin B, 0.5% Noggin, 1mM nicotinamide, 0.5mM N-acetylcysteine, 8. Mu.M ROCK inhibitor, 500nM A83-01, 25ng/ml FGF7, 1. Mu.M SB202190, 500. Mu.g/ml EGF, 250. Mu.g/ml hydrocortisone, 1% insulin transferrin selenium ITS, μm Jagged-1, 1. Mu.MPGE 2 and 2. Mu.M MHY1485.

The preparation method of the cervical squamous carcinoma organoid culture medium adopts the preparation method described in the embodiment 1.

Example 4

The embodiment provides a cervical squamous carcinoma organoid culture medium, which comprises the following components:

DMEM/F12, 12mM HEPES buffer, 1.5 XGlutamax, 1% N2, 1.5 XB 27, 1.5% penicillin-streptomycin, 1.5% amphotericin B, 1% Noggin, 2.5mM nicotinamide, 1.25mM N-acetylcysteine, 12. Mu.M ROCK inhibitor, 500nM A83-01, 25ng/ml FGF7, 1. Mu.M SB202190, 500. Mu.g/ml EGF, 250. Mu.g/ml hydrocortisone, 1% insulin transferrin selenium ITS,. Mu.M Jagged-1, 1. Mu.M PGE2 and 2. Mu.M MHY1485.

The preparation method of the cervical squamous carcinoma organoid culture medium adopts the preparation method described in the embodiment 1.

Example 5

The embodiment provides a cervical squamous carcinoma organoid culture medium, which comprises the following components:

DMEM, 10mM HEPES buffer, 1 XGlutamax, 1% N2, 1 XB 27, 1% penicillin-streptomycin, 1% amphotericin B, 1% Noggin, 2.5mM nicotinamide, 1.25mM N-acetylcysteine, 10. Mu.M ROCK inhibitor, 500nM A83-01, 25ng/ml FGF7, 1. Mu.M SB202190, 500. Mu.g/ml EGF, 250. Mu.g/ml hydrocortisone, 1% insulin transferrin selenium ITS, 1. Mu.M Jagged-1, 1. Mu.MPGE 2, 2. Mu.M MHY1485, 1% L-glutamic acid and 1% L-alanine.

The preparation method of the cervical squamous carcinoma organoid culture medium adopts the preparation method described in the embodiment 1.

Example 6

The embodiment provides a method for culturing cervical squamous carcinoma organoids, which comprises the following steps:

(1) Collection of tumor tissue samples: collecting tumor tissue (obtained from biopsy or surgical specimens) of a patient with primary cervical squamous cell carcinoma, and selecting fresh tumor tissue with slightly tough texture and pink color;

(2) Preservation of tumor tissue samples: immediately after tumor tissue is isolated, the tumor tissue is put into a sample preservation solution, and the sample preservation solution comprises the following components: DMEM/F12, 1% penicillin-streptomycin, 1% amphotericin B; under the ice-cube preservation condition, the ice-cubes are quickly transported to a laboratory;

(3) Washing of the sample: repeatedly cleaning tumor tissues by using precooled calcium-magnesium ion-free PBS to remove mucus and blood stains on the surface of a sample; the vagina is communicated with the outside, so that the vagina contains various miscellaneous bacteria, and the pollution incidence rate of organoid culture can be reduced by repeated cleaning;

(4) Shearing: placing the washed cervical squamous carcinoma tissue on sterile dry gauze to remove excessive water, placing the tumor tissue in a culture dish, and shearing the tumor tissue by using sterile scissors until the tumor tissue is in a fine particle shape and the size is about 1-3mm ³;

(5) Preparing and digesting digestive juice: firstly preparing type IV collagenase powder into collagenase solution with the concentration of 1mg/ml by using DMEM, adding EDTA to make the final concentration of the collagenase solution be 30nM, preparing mixed digestion solution, and preheating the mixed digestion solution in a water bath at 37 ℃; then, the mixed digestive juice (about 5 ml) is used for collecting the tumor tissues in a 15ml centrifuge tube, and the tumor tissues are digested for 10-15 minutes under the water bath condition of 37 ℃;

(6) Collecting the supernatant: standing the centrifuge tube for 1 minute, collecting the supernatant in another centrifuge tube by using a pipetting gun, and stopping digestion by using a serum-free culture solution;

(7) And (5) digesting again: adding about 5ml of mixed digestive juice to the rest tumor tissue precipitate again, digesting for 10-15 min, collecting supernatant, and stopping digestion with serum-free culture solution;

(8) The supernatant was digested and collected: repeating the steps (6) and (7) until the tumor tissue sediment appears as mucus floc, and repeating the whole process for a plurality of times, wherein the whole process is determined according to the size and the texture of the tumor tissue.

(9) And (3) centrifuging: centrifuging the supernatant obtained by repeated collection in a 50ml centrifuge tube for 5 minutes at 300g to obtain a precipitate;

(10) And (5) resuspension: after the supernatant is poured, a sterile cotton swab is used for wiping the wall of the centrifugal tube to prevent backflow, melted Matrigel on ice is added for resuspension, the volume ratio of cell sediment to Matrigel is 1:2, and the operation is carried out on ice;

(11) Inoculating and culturing: inoculating 25 μl/well to a preheated 24-well plate, placing in a 37 ℃ and 5% CO ₂ cell incubator, incubating for 20 min, and after Matrigel is solidified, respectively and independently adding the cervical squamous carcinoma organoid culture medium prepared in the examples 1-2 to culture organoids, and changing liquid once every 2 days to finally obtain the cervical squamous carcinoma organoids. The specific isolated culture flow of cervical squamous carcinoma organoids is shown in FIG. 1.

According to the culturing method of example 6, the cervical squamous carcinoma organoids obtained by culturing the cervical squamous carcinoma organoids prepared in examples 1-2 are similar in morphology as shown in FIG. 2A when observed under an optical microscope, and the cervical squamous carcinoma organoids cultured in the two culture media are in spherical structures and have diameters of more than 200 mu m.

Comparative example 1

This comparative example provides a cervical squamous carcinoma organoid medium that differs from example 1 in that: EGF, hydrocortisone, N2, insulin transferrin selenium ITS, jagged-1, PGE2 and MHY1485 were removed from the medium described in this comparative example, the remainder being the same as in example 1.

According to the culture method of example 6, the cervical squamous carcinoma organoid prepared in this comparative example was cultured with the organoid having a diameter of 80 μm or more, as shown in FIG. 3 (C-E), at 10 days of the culture, the organoid formation efficiency of this comparative example was lower than that of the cervical squamous carcinoma organoid cultured in example 1, and the organoid ratio having a diameter of > 200 μm was significantly lower than that of the cervical squamous carcinoma organoid cultured in example 1.

Comparative example 2

This comparative example explored the effect of insulin transferrin selenium ITS, jagged-1, PGE2 and MHY1485 components on the efficiency of cervical squamous carcinoma organoids in the medium, and compared to the cervical squamous carcinoma organoid medium described in example 1, this comparative example was set up with the medium prepared identically to example 1 lacking insulin transferrin selenium ITS, jagged-1, PGE2 and MHY1485, respectively, and the remainder was cultured according to the culture method described in example 6, as shown in fig. 3F, where "-" in fig. 3F represents the subtraction of the corresponding factors from the medium of comparative example 1 and "+" represents the addition of the corresponding factors from the medium of comparative example 1.

The results show that after the culture medium prepared by the four components is respectively lacking in the culture medium for 10 days, the organoid formation efficiency of the obtained organoid with the diameter more than or equal to 80 mu m is lower than that of the cervical squamous carcinoma organoid cultured in the example 1, and the importance of specific active factors such as insulin transferrin selenium ITS, jagged-1, PGE2, MHY1485 and the like in the culture medium is shown, and the components are matched with each other, so that the metabolic activity and proliferation of cervical cancer cells can be promoted to the greatest extent, and the formation and growth of the cervical squamous carcinoma organoid can be effectively promoted.

Comparative example 3

This comparative example provides a method for culturing cervical squamous carcinoma organoids, which differs from example 6in that: in the step (5), digestion is carried out with only 1mg/ml type IV collagenase, EDTA is not added, and digestion is carried out for 30-60 minutes.

Then, organoid isolated culture was performed using the same mass of cervical squamous carcinoma tumor tissue according to the culture method described in example 6 and this comparative example, using the organoid culture medium prepared in example 1. On day 10 of culture, the number of organoids having a diameter of 80 μm or more in example 6 was significantly larger than that of the cervical squamous carcinoma organoids isolated and cultured in this comparative example, and the results are shown in FIG. 3B; and the formation of the organoid is observed on the 10 th day, the result shows that the proliferation activity (figure 3G) and success rate of the cervical squamous carcinoma organoid cultured by the culture method described in the embodiment 6 are obviously higher than those of the culture method described in the comparative example (table 1), and the results show that the mixed solution of IV collagenase and EDTA is adopted for digestion and collection of cervical squamous carcinoma tumor cells, so that more dispersed single tumor cells and tumor cell clusters can be obtained, and the separation efficiency and the utilization rate of specimens are obviously improved, so that a larger number of tumor organoids are obtained after the culture medium is used for culturing, and the success rate and the formation efficiency of the cervical squamous carcinoma organoid culture are obviously improved.

TABLE 1 success rate of cervical squamous carcinoma organoid culture

Effect example 1

This example was carried out by subjecting the cervical squamous carcinoma organoids obtained by culturing the culture mediums described in examples 1-2, respectively, to HE staining and immunohistochemical staining according to the culture method described in example 6, to observe the structural and phenotypic characteristics of the cultured cervical squamous carcinoma tissue. The specific method is as follows.

Collecting cervical squamous carcinoma organoids in an EP tube, and slicing after agar pre-embedding and paraffin embedding; dewaxing in xylene solution after baking the slices, and then carrying out hematoxylin (3 minutes) and eosin staining (1 minute); for immunohistochemical staining, antigen repair is carried out after baking and dewaxing, then blocking is carried out for 15 minutes by using a blocking agent, and blocking is carried out for 1 hour by using BSA; incubation is carried out by using primary antibody KI67 and P16, overnight at 4 ℃, and DBA color development is carried out after incubation for 30 minutes by using secondary antibody; and the transparent and neutral resin is used for sealing the film, and then photographing can be carried out.

As shown in FIG. 2B, the cervical squamous carcinoma organoid cells were arranged in a disordered manner, had their polarity deleted, and had both the cell proliferation marker KI67 and the HPV persistent infection marker p16.sup.INK4α positive, consistent with the structural and phenotypic characteristics of the cervical squamous carcinoma tissue.

Effect example 2

This effect example was carried out by culturing cervical squamous carcinoma organoids according to the culture medium prepared in example 1 and the culture method described in example 6 to investigate the conditions of passage, cryopreservation and maintenance rate after resuscitation, as follows.

(1) And (3) passage: the cervical squamous carcinoma organoids cultured according to the methods described in example 1 and example 6 were subcultured at least about day 5 of culture. Firstly, using Cell recovery solution solution to dissolve Matrigel on ice for 15-30 min, centrifuging 300g for 5min, and collecting cervical squamous carcinoma organoids in a centrifuge tube; the organoid is cracked by adopting a mechanical digestion method, about 500 mu l of organoid culture solution is added into organoid sediment, 200 mu l of gun heads are used for repeatedly blowing, the organoid is broken into cell clumps with different sizes, and the cell clumps are centrifuged after being resuspended, so that sediment is obtained; matrigel was added to resuspended (on ice) at a 1:2 ratio of cell pellet to Matrigel volume; after solidification, the cervical squamous carcinoma organoid culture solution prepared in the example 1 can be added for subculture.

(2) Freezing: mechanically digesting according to the method to obtain organoid precipitate, and adding serum-free frozen stock solution for resuspension; then transferring into a program cooling box, placing in a refrigerator at-80 ℃ for overnight, and finally transferring into a liquid nitrogen tank for long-term freezing storage.

(3) Resuscitating: taking out the frozen cervical squamous carcinoma organoids from the liquid nitrogen tank, and immediately placing the frozen cervical squamous carcinoma organoids in a water bath at 37 ℃ for melting; adding the organoid culture solution according to the volume ratio of 1:9, uniformly mixing, centrifuging to obtain a precipitate, adding Matrigel for resuspension, solidifying, and adding the organoid culture solution for culture.

The cervical squamous carcinoma organoids obtained by subculture and resuscitating culture after freezing are observed under an optical microscope as shown in the graph (C-D) in fig. 2, and the phenotypic characteristics of the cervical squamous carcinoma organoids recovered after the culture is transferred to the third generation, the ninth generation and the freezing are similar to those of the cervical squamous carcinoma organoids of the first generation, and are in a spherical structure, the diameter of the cervical squamous carcinoma organoids can still reach more than 200 mu m, so that the cervical squamous carcinoma organoids cultured by the culture medium have good maintenance rate after the subculture and can support long-term maintenance.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (9)

1. A cervical squamous carcinoma organoid medium comprising the following components: basal medium, HEPES buffer, nutritional additives and specific active factors;

The specific active factors comprise penicillin-streptomycin, amphotericin B, noggin, nicotinamide, N-acetylcysteine, ROCK inhibitor, A83-01, FGF7, SB202190, EGF, hydrocortisone, insulin transferrin selenium ITS, jagged-1, PGE2 and MHY1485;

the nutritional additives include Glutamax, N2 and B27.

2. The cervical squamous carcinoma organoid medium of claim 1, comprising the following components: basal medium, 8-12mM HEPES buffer, 0.5-1.5 XGlutamax, 0.5-1% N2, 0.5-1.5XB27, 0.5-1.5% penicillin-streptomycin, 0.5-1.5% amphotericin B, 0.5-1% noggin, 1-2.5mM nicotinamide, 0.5-1.25mM N-acetylcysteine, 8-12. Mu.M ROCK inhibitor, 200-500nM A83-01, 10-25ng/ml FGF7, 0.1-1. Mu.M SB202190, 200-500. Mu.g/ml EGF, 100-250. Mu.g/ml hydrocortisone, 0.5-1% insulin transferrin selenium ITS, 0.1-1. Mu.M Jagged-1, 0.1-1. Mu.M PGE2 and 0.5-2. Mu. MMHY.

3. The cervical squamous carcinoma organoid medium of claim 2, comprising the following components: basal medium, 10mM HEPES buffer, 1 XGlutamax, 1% N2, 1 XB 27, 1% penicillin-streptomycin, 1% amphotericin B, 1% noggin, 2.5mM nicotinamide, 1.25mM N-acetylcysteine, 10. Mu.M ROCK inhibitor, 500nM A83-01, 25ng/ml FGF7, 1. Mu.M SB202190, 500. Mu.g/ml EGF, 250. Mu.g/ml hydrocortisone, 1% insulin transferrin selenium ITS,. Mu.M Jagged-1, 1. Mu.MPGE 2 and 2. Mu.M MHY1485.

4. The cervical squamous carcinoma organoid medium of claim 1, wherein the basal medium is DMEM or DMEM/F12.

5. The cervical squamous carcinoma organoid medium of claim 1, further comprising the following components: 0.5-1.5% L-glutamic acid, 0.5-1.5% L-alanine or 0.5-1.5% glycine.

6. A method for culturing cervical squamous carcinoma organoids, comprising the steps of:

(1) Collecting cervical squamous cell carcinoma tissue, cleaning to remove blood clots and mucus, and shearing the carcinoma tissue into fine particles;

(2) Adding mixed solution containing IV collagenase and EDTA into sheared cancer tissue, shaking table digestion for 10-15min, standing for 1-3min, collecting supernatant, and stopping digestion with serum-free culture solution;

(3) Repeating the digestion and standing process of the step (2) on the rest cancer tissue sediment, collecting the supernatant and stopping the digestion until the cancer tissue appears as mucus flocculent;

(4) Centrifuging the repeatedly collected supernatant to obtain a cell precipitate, adding Matrigel for resuspension, inoculating, fixing, adding the cervical squamous carcinoma organoid culture medium according to any of claims 1-5, and culturing to obtain the cervical squamous carcinoma organoid.

7. The method according to claim 6, wherein in the step (2), the concentration of type IV collagenase in the mixed solution is 0.5-1mg/ml, and the concentration of EDTA is 30-50nM.

8. The method according to claim 6, wherein in the step (4), the volume ratio of the cell pellet to Matrigel is 1:2 to 1:3.

9. Use of the medium according to any one of claims 1-5 for culturing cervical squamous carcinoma organoids.