CN114438032B - Composition, culture medium and method for 3D culture of laryngeal cancer tissues - Google Patents

Abstract

The invention provides a composition, a culture medium and a method for 3D culture of laryngeal cancer tissues. The composition of the medium according to final concentration comprises: b27 serum-free additive, 0.5-2 ×; wnt3A,50-500ng/ml; n-acetylysteine,0.15 to 1.5mM; EGF,20 to 100ng/mL; noggin,50 to 200ng/mL; r-spondin 1, 200 to 1000ng/mL; GSK1838705A,0.2-2nM; 10 to 50ng/mL of FGF 10; nicotinamide,5 to 20mM; y-27632 at 1-20 μ M; the solvent is SAGM culture medium. The organoid formed by the invention can maintain the tissue cell specificity, the stem cell characteristic and the genotyping highly consistent, and the cell morphology and physiological functions are highly similar.

Description

A composition, medium and method for 3D culture of laryngeal cancer tissue

technical field

The invention relates to the technical field of organoid culture, in particular to a composition, medium and method for 3D culture of laryngeal cancer tissue.

Background technique

According to the latest 2018 global cancer report released by the International Agency for Research on Cancer (IARC) under the World Health Organization, China ranks first in the world in cancer incidence and mortality. Data show that there are 3.804 million new cancer cases and 2.296 million cancer deaths in China, which is equivalent to more than 10,000 people being diagnosed with cancer and more than 6,000 people dying of cancer every day in my country. Cancer treatment is one of the biggest and most urgent problems in the field of biomedicine. On the one hand, there are more and more laboratory researches, on the other hand, the efficiency of clinical transformation of new drugs is still low. Organoid culture provides a new technical platform for the rapid and effective development of tumor drugs.

The preservation of the heterogeneity of the source tumor tissue by tumor organoids is the core basis of organoid research. Studies have found that a large number of tumor organoids with different characteristics can be obtained by in vitro organoid culture of tumor tissue, and the analysis results of a single organoid also show the heterogeneity of organoids from the same tumor source. At the same time, histochemical analysis found that there was a tissue structure similar to that of the source tumor inside the tumor organoid, and in situ DNA analysis further confirmed that the same gene mutation site as the source tumor also existed in the organoid. It can be seen that tumor organoids reproduce the diversity and complexity of their source tumors at a high level in terms of genes, transcription, metabolism, cells and histology. More importantly, the in vitro culture process does not appear to be significantly homogenous to tumor organoids. Compared with traditional 2D culture and tumor tissue xenografting, on the one hand, the success rate of tumor organoid construction is significantly higher, and it can be cultured quickly and at low cost for a long time, which is convenient for gene modification and large-scale drug screening; on the other hand, 3D culture retains tumor In the research process, the influence of the tumor microenvironment will not be lost, providing a more realistic environment for the development of tumor drugs. At present, tumor organoids from various tissues including colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, gastric cancer, etc. have been successfully constructed, but there is no research report on the culture of laryngeal cancer tissue organoids.

Laryngeal cancer is one of many cancer types that affect the head and neck area. It affects: the area of the throat behind the nose - the nasopharynx; the middle of the throat - the oropharynx; the bottom of the throat - the hypopharynx; the mouth, where the tongue is located; and the larynx, which is needed for speech. Cancers arising at this site are complex and painful, but most (65%-80%) patients survive. At present, ordinary culture techniques are mainly used for laryngeal cancer tissues. In the two-dimensional culture process, laryngeal cancer tissue cells are difficult or unable to fully express the pathophysiological characteristics of laryngeal cancer, so that the cultured primary laryngeal cancer cells and living laryngeal cancer The cells are quite different, making it impossible to carry out basic and clinical research on laryngeal cancer. In 3D culture, if a suitable medium cannot be found, it is difficult to grow organoids from laryngeal cancer tissues. The present invention aims to fill the research gap in this regard.

Contents of the invention

Aiming at the current blank of laryngeal cancer organoid culture technology in China, the present invention provides a composition, culture medium and method for 3D culture of laryngeal cancer tissue. Technical scheme of the present invention is:

In the first aspect, the present invention provides a composition for 3D culture of laryngeal cancer tissue, including: B27 serum-free supplement, Wnt signaling pathway protein, N-acetylsteine, epidermal growth factor, BMP antagonist, R-spondin 1 , IGF-1R inhibitors, fibroblast growth factors, Nicotinamide, selective inhibitors of ROCK-related protein kinases.

Further, the composition also includes: p38 MAPK inhibitor.

Further, the composition at least includes: B27 serum-free supplement, Wnt3A, N-acetylsteine, EGF, Noggin, R-spondin 1, GSK1838705A, FGF10, Nicotinamide, Y-27632.

Preferably, the composition further includes: at least one of FGF2 and SB202190. Optionally, the composition further includes: at least one of trans-retinoic acid and BMP7.

In the second aspect, the present invention provides a medium for 3D culture of laryngeal cancer tissue, which comprises: B27 serum-free supplement, 0.5-2×; Wnt3A, 50-500ng/ml; N-acetylsteine, according to the composition of the final concentration. 0.15~1.5mM; EGF, 20~100ng/mL; Noggin, 50~200ng/mL; R-spondin 1, 200~1000ng/mL; GSK1838705A, 0.2-2nM; FGF10, 10~50ng/mL; Nicotinamide, 5~ 20mM; Y-27632, 1~20μM; solvent is SAGM medium.

Preferably, the medium further includes at least one of the following according to the composition of the final concentration: FGF2, 5-50 ng/mL; SB202190, 5-15 μM.

Optionally, the medium further includes at least one of the following according to the composition of the final concentration: trans-retinoic acid: 20-200 nM; BMP7: 2-10 ng/mL.

In a third aspect, the present invention provides a method for 3D culture of laryngeal cancer tissue, comprising the following steps:

Step 1: resuspend the shredded laryngeal cancer tissue in digestive solution I and perform a digestion, and remove the digestive solution I after digestion;

Step 2: Add Digestion Solution II to the system to resuspend and perform secondary digestion. After digestion, remove the precipitate and retain the supernatant;

Step 3: Add DMEM/F12 to the supernatant, mix well, pass through a 100 μm sieve and centrifuge to retain the cell pellet;

Step 4: Add red blood cell lysate to the cell pellet to resuspend, then remove the supernatant, add DMEM/F12 to resuspend, and remove the supernatant again;

Step 5: Mix the cells and matrigel according to the ratio of 30000 cells per 30 μL, drop them into the well plate, and add the above-mentioned medium, and culture at 37°C and 5% CO ₂ for 10-14 days, with intervals of 3- The medium was changed every 4 days.

Further, the digestion solution I is a DMEM medium containing 200 U/ml collagenase III and 5 mg/ml dispase II.

Further, the digestion solution II is a DMEM medium containing 100 U/ml hyaluronidase and 0.1 mg/ml Dnase I.

Compared with prior art, the beneficial effect of the present invention is:

The culture medium of laryngeal cancer tissue organoids of the present invention is aimed at the culture and growth characteristics of laryngeal cancer tissue-derived cells. A variety of cytokine components are selected and formulated according to a certain ratio. Laryngeal cancer cells can effectively form organoids in the 3D medium. Organs, the formed organoids can maintain tissue cell specificity, stem cell characteristics, and genotyping are highly consistent, cell morphology, and physiological functions are also highly similar. In addition, the culture medium of the present invention can complete the cryopreservation, recovery, and passage of laryngeal cancer organoids, meet the requirements of large-scale replication of laryngeal cancer organoids, and control the cultured laryngeal cancer organoids to have a high degree of consistency.

Description of drawings

Fig. 1 is a diagram showing the morphology and structure of laryngeal cancer organoids obtained in Example 6 of the present invention.

Fig. 2 is a diagram of the morphology and structure of laryngeal cancer organoids obtained in Example 7 of the present invention.

Fig. 3 is a diagram showing the morphology and structure of laryngeal cancer organoids obtained in Example 8 of the present invention.

Fig. 4 is a diagram showing the morphology and structure of laryngeal cancer organoids obtained in Example 9 of the present invention.

Fig. 5 is a diagram showing the morphology and structure of laryngeal cancer organoids obtained in Example 10 of the present invention.

Fig. 6 is a morphological structure diagram of laryngeal cancer organoids of six passages in Example 11 of the present invention.

Fig. 7 is a morphological diagram of laryngeal cancer organoids after cryopreservation and resuscitation in Example 12 of the present invention.

FIG. 8 is a morphological diagram of laryngeal cancer organoids obtained in Comparative Example 1 of the present invention.

FIG. 9 is a morphological diagram of laryngeal cancer organoids obtained in Comparative Example 2 of the present invention.

FIG. 10 is a morphological diagram of laryngeal cancer organoids obtained in Comparative Example 3 of the present invention.

Detailed ways

In the description of the present invention, it should be noted that those in the examples that do not specify specific conditions shall be carried out according to conventional conditions or conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

A specific embodiment of the present invention provides a composition for 3D culture of laryngeal cancer tissue, by adding B27 serum-free additive, Wnt signaling pathway protein, N-acetylsteine, epidermal growth factor, BMP antagonist, R-spondin1, IGF -1R inhibitor, fibroblast growth factor, Nicotinamide, ROCK-related protein kinase selective inhibitor, p38 MAPK inhibitor and other components were compounded and mixed with an appropriate basal medium to obtain a tissue suitable for laryngeal cancer 3D culture medium, laryngeal cancer cells can effectively form organoids in this 3D medium, and the formed organoids can maintain tissue cell specificity, stem cell characteristics, genotyping highly consistent, cell morphology, and physiological functions are also highly similar . In the foregoing composition, at least include: B27 serum-free supplement, Wnt3A, N-acetylsteine, EGF, Noggin, R-spondin 1, GSK1838705A, FGF10, Nicotinamide, Y-27632.

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments, which are explanations rather than limitations of the present invention.

Example 1

The invention provides a medium for 3D culture of laryngeal cancer tissue, which comprises according to the final concentration: B27 serum-free additive, 0.5×; Wnt3A, 200ng/ml; N-acetylsteine, 1.5mM; EGF, 30ng/mL; Noggin, 150ng/mL; R-spondin 1, 200ng/mL; GSK1838705A, 2nM; FGF10, 40 ng/mL; Nicotinamide, 20mM; Y-27632, 2μM; SB202190, 10μM; ng/mL. The solvent is SAGM medium. The preparation method of the culture medium is as follows: compose the ingredients according to the final concentration of all the components, and then use the SAGM culture medium to mix evenly.

Example 2

The invention provides a culture medium for laryngeal cancer tissue 3D culture, which comprises according to the final concentration: B27 serum-free additive, 2×; Wnt3A, 50ng/ml; N-acetylsteine, 1mM; EGF, 100ng/mL; Noggin , 200ng/mL; R-spondin 1, 1000ng/mL; GSK1838705A, 2nM; FGF10, 10ng/mL; Nicotinamide, 5mM; Y-27632, 20μM; FGF2, 30ng/mL; . The solvent is SAGM medium. The preparation method of the culture medium is the same as in Example 1.

Example 3

The invention provides a medium for 3D culture of laryngeal cancer tissue, which comprises: B27 serum-free additive, 1×; Wnt3A, 500ng/ml; N-acetylsteine, 0.15mM; EGF, 60ng/mL according to the composition of the final concentration; Noggin, 50ng/mL; R-spondin 1, 500ng/mL; GSK1838705A, 0.5nM; FGF10, 50ng/mL; Nicotinamide, 10mM; Y-27632, 10μM; FGF2, 30ng/mL; : 100nM; BMP7: 10 ng/mL.

The solvent is SAGM medium. The preparation method of this medium is the same as that in Example 1.

Example 4

The invention provides a medium for 3D culture of laryngeal cancer tissue, which comprises according to the final concentration: B27 serum-free additive, 1.5×; Wnt3A, 300ng/ml; N-acetylsteine, 0.5mM; EGF, 20ng/mL; Noggin, 100ng/mL; R-spondin 1, 700ng/mL; GSK1838705A, 0.2nM; FGF10, 30ng/mL; Nicotinamide, 15mM; Y-27632, 5μM; FGF2, 10ng/mL; /mL.

The solvent is SAGM medium. The preparation method of this medium is the same as that in Example 1.

Example 5

The invention provides a culture medium for laryngeal cancer tissue 3D culture, which comprises according to the final concentration: B27 serum-free additive, 0.3×; Wnt3A, 80ng/ml; N-acetylsteine, 1.2mM; EGF, 50ng/mL; Noggin, 150ng/mL; R-spondin 1, 300ng/mL; GSK1838705A, 0.5nM; FGF10, 20ng/mL; Nicotinamide, 8mM; Y-27632, 15μM; FGF2, 50ng/mL; : 100nM; BMP7: 5ng/mL; solvent is SAGM medium. The preparation method of this medium is the same as that in Example 1.

Example 6

This embodiment provides a method for 3D culture of laryngeal cancer tissue, comprising the following steps:

Step 1: Take laryngeal cancer tissue and cut it into pieces on ice, add 5ml collagenase III + cell dispase II mixed digestion solution I to resuspend, transfer to 37°C, 220rpm shaker for digestion for 150min, and centrifuge at 1500rpm to remove digestion solution I. The final concentrations of collagenase III and dispase II in digestion solution I were 200 U/ml and 5 mg/ml, respectively.

Step 2, add 5ml of hyaluronidase + DNase I mixed digestion solution II to the system again, the final concentrations of hyaluronidase and DNase I in the digestion solution II are 100U/ml and 0.1mg/ml respectively, at 37°C , 220rpm shaker digestion for 5min, after the digestion, remove the precipitate and retain the supernatant.

Step 3: Add DMEM/F12 to the supernatant, mix well, pass through a 100 μm sieve, and centrifuge at 4°C, 1200 rpm for 3 minutes to retain the cell pellet;

Step 4: Add erythrocyte lysate to the cell pellet to resuspend the cells for 5 minutes, then centrifuge at 4°C, 1200rpm for 3min, remove the supernatant, add DMEM/F12 to resuspend, centrifuge at 4°C, 1200rpm for 3min, remove the supernatant again;

Step 5, cell counting, mix cells and matrigel according to the ratio of 30000 cells per 30 μL, drop into the well plate, drop in the center of the well of the 6-well plate, 3 glue drops per well, put the culture dish at 37 ℃, 5% Place it under CO ₂ for 10 minutes to solidify the Marteigel. Add 3mL of the medium of Example 1 to each well, and culture at 37°C and 5% _CO for 14 days, during which the medium was replaced every 3-4 days. The laryngeal cancer organoids obtained in this example are shown in Figure 1.

Example 7

This example provides a method for 3D culture of laryngeal cancer tissue, the process is the same as that of Example 6, and the culture medium of Example 2 is used. The obtained laryngeal cancer organoids are shown in Figure 2.

Example 8

This example provides a method for 3D culture of laryngeal cancer tissue, the process is the same as that of Example 6, and the culture medium of Example 3 is used. The obtained laryngeal cancer organoids are shown in Figure 3.

Example 9

This example provides a method for 3D culture of laryngeal cancer tissue, the process is the same as that of Example 6, and the culture medium of Example 4 is used. The obtained laryngeal cancer organoids are shown in Figure 4.

Example 10

This example provides a method for 3D culture of laryngeal cancer tissue, the process is the same as that of Example 6, the culture medium of Example 5 is used, and the culture time is 10 days. The obtained laryngeal cancer organoids are shown in Figure 5.

Example 11

Multiple passages of laryngeal cancer organoids

After the primary laryngeal cancer organoids were obtained according to Example 8, they were subcultured, and the subculture operation was as follows:

1. Use a pipette to suck out the culture medium in the culture dish, take 1-2 ml TrypLE™ Express to digest the gel drop containing organoids, put it in the incubator, and digest at 37°C for 10 min;

2. Add 10 ml of sterile Hank’s balanced salt solution, centrifuge at 1000 rpm for 3 minutes;

3. Discard the supernatant, add 200 μl of the medium of Example 1 to resuspend the organoids, add 250 μl of Matrigel to mix well, drop into a new 35 mm culture dish, let it stand for 5 minutes, transfer it into the incubator, turn it upside down, After 40 min, add 2-4 ml of the medium of Example 3 and culture in a 37°C incubator.

After 10 days of subculture, the second-generation laryngeal cancer organoids were obtained. According to this method, the second-generation laryngeal cancer organoids were obtained. After 45 days of culture, the sixth-generation laryngeal cancer organoids were obtained. The shape of the organ structure is good, and the laryngeal cancer organoids after 6 passages can still maintain the morphology of the original tissue.

Example 12

Cryopreservation and recovery of laryngeal cancer organoids

After the primary laryngeal cancer organoids were obtained according to Example 8, they were cryopreserved, and the cryopreservation operation was as follows:

Use a pipette to suck out the culture medium in the culture dish, take 1-2 ml of cell recovery solution to digest the gel droplets containing organoids at room temperature for 5-10 min, and repeatedly blow with a pipette to blow the gel droplets; add 10 ml Sterile Hank's balanced salt solution, 1000rpm, centrifuge for 3min; discard the supernatant, add 1ml freezing solution (90%FBS + 10%DMSO) to resuspend organoids, transfer to cryopreservation tube, program cooling (4℃ →-20℃→-80℃→liquid nitrogen), and finally frozen in liquid nitrogen. It can also be stored in a -80°C refrigerator.

The procedure for resuscitating cryopreserved laryngeal cancer organoids is as follows:

(1) Take out the cryopreservation tube, immerse it in warm water at 37°C directly, and shake it from time to time to melt it as soon as possible.

(2) Take out the cryopreservation tube from the 37°C water bath, move it to a biological safety cabinet, open the lid, suck out the cell suspension with a straw, add it to the centrifuge tube and drop more than 10 times of SAGM medium, and mix well. Centrifuge at 1000 rpm for 3 min.

(3) Discard the supernatant, add 120 μl of the medium of Example 3 to resuspend the organoids, add 150 μl of Matrigel to mix well, drop into a 35 mm culture dish, let it stand for 5 minutes, transfer it into the incubator, turn it upside down, and after 40 minutes , supplemented with 2-4 ml of the medium of Example 3, and cultured in a 37°C incubator.

(4) The culture medium was replaced once the next day, and laryngeal cancer organoids were obtained after continuing to culture for 4 days, as shown in Figure 7.

Comparative example 1

This comparative example provides direct use of the existing medium commonly used for organoid culture for laryngeal cancer organoid culture, and the operation is as follows:

Ordinary medium (DMEM+10%FBS) was used, and the operation process was the same as in Example 6. As a result, the cells of laryngeal cancer cells adhered to the bottom of the culture dish during the culture process, which was similar to the results of general cell culture, and could not form structured, multi- Organoids of cellular components. Figure 8 provides a diagram of the morphology and structure of organoids cultured for 14 days in this comparative example.

Comparative example 2

This comparative example provides a laryngeal cancer medium, which is different from Example 1 in that EGF is not added, and the method for culturing laryngeal cancer organoids using this laryngeal cancer medium is the same as that in Example 6. Results The formation process of laryngeal cancer organoids was extremely slow, the number was small, and the diameter was small. Figure 9 provides the morphological structure diagram of organoids cultured for 14 days in this comparative example.

Comparative example 3

This comparative example provides a laryngeal cancer medium, which differs from Example 1 in that R-spondin 1 is not added, and the method for culturing laryngeal cancer organoids using this laryngeal cancer medium is the same as that in Example 6. Results The laryngeal cancer organoids formed slowly and grew slowly. Figure 10 provides a morphological structure diagram of the organoids cultured for 14 days in this comparative example.

In summary, the organoids formed by using the laryngeal cancer tissue organoid culture medium of the present invention can maintain a high degree of consistency in tissue cell specificity, stem cell characteristics, and genotyping, and the cell morphology and physiological functions are also highly similar. Moreover, the culture medium of the present invention can complete the cryopreservation, recovery, and passage of laryngeal cancer organoids, meet the demand for large-scale replication of laryngeal cancer organoids, and control the cultured laryngeal cancer organoids to have a high degree of consistency.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (3)

1. A culture medium for 3D culture of laryngeal cancer tissue, comprising: the composition according to final concentration is: b27 serum-free additive, 0.5-2 ×; wnt3A,50-500ng/ml; n-acetylysteine,0.15 to 1.5mM; EGF,20 to 100ng/mL; noggin,50 to 200ng/mL; r-spondin 1, 200 to 1000ng/mL; GSK1838705A,0.2-2nM; 10 to 50ng/mL of FGF 10; nicotinamide,5 to 20mM; y-27632, 1-20 mu M, trans-retinoic acid, 20-200nM; BMP7,2 to 10ng/mL; FGF2,5 to 50ng/mL; SB202190,5 to 15 μ M; the solvent is SAGM culture medium.

2. A method for 3D culture of laryngeal cancer tissue, characterized in that: the method comprises the following steps:

the method comprises the following steps of firstly, carrying out primary digestion after resuspending the cut laryngeal cancer tissues by adopting a digestive juice I, and removing the digestive juice I after the digestion is finished;

secondly, adding the digestive juice II into the system again, performing secondary digestion after resuspension, and removing precipitates and reserving clear liquid after digestion is finished;

adding DMEM/F12 into the clear liquid, mixing uniformly, passing through a 100-micron screen, centrifuging and then retaining cell sediment;

adding erythrocyte lysate into the cell sediment for resuspension, then removing the supernatant, adding DMEM/F12 for resuspension again, and removing the supernatant again;

step five, mixing the cells and matrigel at a ratio of 30000 cells per 30. Mu.L, dropping the mixture into a well plate, adding the medium according to claim 1, and 5% CO at 37 ℃ ₂ Culturing for 10 to 14 days under the condition of (1), and replacing the culture medium every 3 to 4 days.

3. The method for 3D culture of laryngeal cancer tissue according to claim 2, wherein: the digestive juice I is a DMEM medium containing 200U/ml collagenase III and 5mg/ml cell dispersing enzyme II; the digestive juice II is a DMEM medium containing 100U/ml hyaluronidase and 0.1mg/ml Dnase I.

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