CN114438032B - Composition, culture medium and method for 3D culture of laryngeal cancer tissues - Google Patents
Composition, culture medium and method for 3D culture of laryngeal cancer tissues Download PDFInfo
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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
Technical Field
The invention relates to the technical field of organoid culture, in particular to a composition, a culture medium and a method for 3D culture of laryngeal cancer tissues.
Background
According to 2018 global Cancer reports newly issued by International Agency for Research on Cancer (IARC) under the flag of the world health organization, the incidence and the mortality of Chinese Cancer are the first worldwide. The data shows that 380.4 thousands of new cancer cases and 229.6 thousands of cancer death cases exist in China, which is equivalent to that more than 1 million people in China have diagnosed cancer and 6000 people die of cancer every day. Tumor therapy is one of the biggest and most urgent problems in the biomedical field at present. On one hand, more and more laboratory researches are carried out, and on the other hand, the clinical conversion efficiency of new drugs is still low. Organoid culture provides a new technical platform for the rapid and effective research and development of tumor drugs.
Preservation of tumor organoids against heterogeneity of source tumor tissue is the core basis of organoid research. Research shows that a large number of tumor organoids with different characteristics can be obtained by culturing the tumor tissues in vitro organoids, and the analysis result of a single organoid also shows the heterogeneity of organoids from the same tumor. Meanwhile, histochemical analysis finds that the tissue structure similar to the original tumor exists in the tumor organoid, and in-situ DNA analysis further proves that the same gene mutation sites of the original tumor exist in the organoid. It follows that tumor organoids reproduce the diversity and complexity of their tumor sources at a high level in terms of gene, transcription, metabolism, cell and histology. More importantly, the in vitro culture process does not appear to be significantly homogeneous for tumor organoids. Compared with the traditional 2D culture and tumor tissue xenotransplantation, the tumor organoid has obviously improved construction success rate, can be rapidly cultured for a long time at low cost, and is convenient for gene modification, large-scale drug screening and the like; on the other hand, the 3D culture maintains the tissue characteristics of the tumor, the influence of the tumor microenvironment can not be lost in the research process, and a more real environment is provided for the research and development of tumor drugs. At present, tumor organoids of various tissues including colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, gastric cancer and the like have been successfully constructed, but research reports on culture of laryngeal cancer tissue organoids are not available.
Laryngeal cancer is one of the many types of cancer that affect the head and neck region. Its range of influence includes: the throat region behind the nose-the nasopharynx; the middle part of the throat-oropharynx; bottom of throat-hypopharynx; the oral cavity in which the tongue is located; and the larynx required for speaking. The cancer that develops at this site is complex and painful, but most (65% -80%) of patients survive. The existing laryngeal cancer tissue mainly adopts a common culture technology, and laryngeal cancer tissue cells are difficult to or cannot fully express the pathophysiology characteristics of laryngeal cancer in a two-dimensional culture process, so that the cultured laryngeal cancer primary cells and living laryngeal cancer cells have larger difference, and the basic and clinical research of laryngeal cancer cannot be carried out. In 3D culture, laryngeal cancer tissues are difficult to culture into organoids if a suitable culture medium cannot be found. The present invention aims to fill the blank of research in this respect.
Disclosure of Invention
Aiming at the blank of the current laryngeal cancer organoid culture technology in China, the invention provides a composition, a culture medium and a method for 3D culture of laryngeal cancer tissues. The technical scheme of the invention is as follows:
in a first aspect, the present invention provides a composition for 3D culture of laryngeal cancer tissue, comprising: b27 serum-free additive, wnt signal path protein, N-acetylserine, epidermal growth factor, BMP antagonist, R-spondin 1, IGF-1R inhibitor, fibroblast growth factor, nicotinamide and ROCK related protein kinase selective inhibitor.
Further, the composition further comprises: p38 MAPK inhibitors.
Further, the composition comprises at least: b27 serum-free additive, wnt3A, N-acetylystein, EGF, noggin, R-spondin 1, GSK1838705A, FGF10, nicotinamide and Y-27632.
Preferably, the composition further comprises: at least one of FGF2 and SB 202190. Optionally, the composition further comprises: at least one of trans-retinoic acid and BMP 7.
In a second aspect, the present invention provides a medium for 3D culture of laryngeal cancer tissue, comprising, in terms of final concentration composition: 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.
Preferably, the composition of the medium in terms of final concentration further comprises at least one of the following: FGF2,5 to 50ng/mL; SB202190,5 to 15. Mu.M.
Optionally, the composition of the medium in terms of final concentration further comprises at least one of: trans-retinoic acid: 20-200nM; BMP7:2 to 10 ng/mL.
In a third aspect, the present invention provides a method for 3D culture of laryngeal cancer tissue, comprising the steps of:
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 a 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 into a well plate, and adding the above medium, at 37 ℃ with 5% CO 2 Culturing for 10 to 14 days under the condition of (1), and replacing the culture medium every 3 to 4 days.
Further, the digestion solution I is a DMEM medium containing 200U/ml of collagenase III and 5mg/ml of cell dispersing enzyme II.
Further, the digestion solution II was DMEM medium containing 100U/ml hyaluronidase and 0.1mg/ml dnase I.
Compared with the prior art, the invention has the beneficial effects that:
the culture medium of the laryngeal cancer tissue organoid is prepared by selecting various cell factor components according to a certain proportion aiming at the culture growth characteristics of laryngeal cancer tissue source cells, the laryngeal cancer cells can effectively form the organoid in the 3D culture medium, and the formed organoid can maintain the tissue cell specificity, the stem cell characteristic and the genotyping height to be consistent, and has highly similar cell morphology and physiological function. In addition, the culture medium can finish cryopreservation, resuscitation and passage of the laryngeal cancer organoid, meet the requirement of large-scale replication of the laryngeal cancer organoid, and control the laryngeal cancer organoid obtained by culture to have high consistency.
Drawings
FIG. 1 is a morphological structural diagram of a laryngeal cancer organoid obtained in example 6 of the present invention.
FIG. 2 is a diagram showing a morphological structure of a laryngeal cancer organoid obtained in example 7 of the present invention.
FIG. 3 is a diagram showing a morphological structure of a laryngeal cancer organoid obtained in example 8 of the present invention.
FIG. 4 is a diagram showing a morphological structure of a laryngeal cancer organoid obtained in example 9 of the present invention.
FIG. 5 is a diagram showing a morphological structure of a laryngeal cancer organoid obtained in example 10 of the present invention.
FIG. 6 is a structural diagram showing the morphology of throat cancer organoids at 6 passages in example 11 of the present invention.
FIG. 7 is a morphological diagram of laryngeal carcinoma organoids after cryopreservation resuscitation according to 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 Description
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a composition for 3D culture of laryngeal cancer tissues, which is prepared by compounding components such as a B27 serum-free additive, wnt signal channel protein, N-acetylystein, epidermal cell growth factor, BMP antagonist, R-spondin 1, IGF-1R inhibitor, fibroblast growth factor, nicotinamide, ROCK-related protein kinase selective inhibitor, p38 MAPK inhibitor and the like, and mixing the components by adopting a proper basic culture medium to obtain a culture medium suitable for 3D culture of the laryngeal cancer tissues. In the composition as described above, at least: b27 serum-free additive, wnt3A, N-acetyllysine, EGF, noggin, R-spondin 1, GSK1838705A, FGF10, nicotinamide and Y-27632.
The invention will be described in further detail with reference to the following figures and specific examples, which are given by way of illustration and not by way of limitation.
Example 1
The invention provides a culture medium for 3D culture of laryngeal cancer tissues, which comprises the following components in final concentration: b27 serum-free additive, 0.5 ×; wnt3A,200ng/ml; n-acetylysteine,1.5mM; EGF,30ng/mL; noggin,150ng/mL; r-spondin 1, 200ng/mL; GSK1838705A,2nM; FGF10, 40 ng/mL; nicotinamide,20mM; y-27632 at 2. Mu.M; SB202190, 10. Mu.M; trans-retinoic acid: 100nM; BMP7:10 ng/mL. The solvent is SAGM culture medium. The preparation method of the culture medium comprises the following steps: preparing ingredients according to the final concentration of all the components, and then uniformly mixing the ingredients with an SAGM culture medium.
Example 2
The invention provides a culture medium for 3D culture of laryngeal cancer tissues, which comprises the following components in final concentration: b27 serum-free additive, 2 ×; wnt3A,50ng/ml; n-acetylystein, 1mM; EGF,100ng/mL; noggin,200ng/mL; r-spondin 1, 1000ng/mL; GSK1838705A,2nM; FGF10, 10ng/mL; nicotinamide,5mM; y-27632, 20. Mu.M; FGF2, 30ng/mL; SB202190, 10. Mu.M; trans-retinoic acid: 100nM. The solvent is SAGM culture medium. The medium was prepared in the same manner as in example 1.
Example 3
The invention provides a culture medium for 3D culture of laryngeal cancer tissues, which comprises the following components in final concentration: b27 serum-free additive, 1 ×; wnt3A,500ng/ml; n-acetylystein, 0.15mM; EGF,60ng/mL; noggin,50ng/mL; r-spondin 1, 500ng/mL; GSK1838705A,0.5nM; FGF10, 50ng/mL; nicotinamide,10mM; y-27632, 10. Mu.M; FGF2, 30ng/mL; SB202190, 10. Mu.M; trans-retinoic acid: 100nM; BMP7:10 ng/mL.
The solvent is SAGM culture medium. The medium was prepared in the same manner as in example 1.
Example 4
The invention provides a culture medium for 3D culture of laryngeal cancer tissues, which comprises the following components in final concentration: b27 serum-free additive, 1.5 ×; wnt3A,300ng/ml; n-acetylystein, 0.5mM; EGF,20ng/mL; noggin,100ng/mL; r-spondin 1, 700ng/mL; GSK1838705A,0.2nM; FGF10, 30ng/mL; nicotinamide,15mM; y-27632 at 5. Mu.M; FGF2, 10ng/mL; SB202190, 15. Mu.M; BMP7:10 ng/mL.
The solvent is SAGM culture medium. The medium was prepared in the same manner as in example 1.
Example 5
The invention provides a culture medium for 3D culture of laryngeal cancer tissues, which comprises the following components in final concentration: b27 serum-free additive, 0.3 ×; wnt3A,80ng/ml; n-acetylysteine,1.2mM; EGF,50ng/mL; noggin,150ng/mL; r-spondin 1, 300ng/mL; GSK1838705A,0.5nM; FGF10, 20ng/mL; nicotinamide,8mM; y-27632, 15. Mu.M; FGF2, 50ng/mL; SB202190, 5. Mu.M; trans-retinoic acid: 100nM; BMP7:5ng/mL; the solvent is SAGM culture medium. The medium was prepared in the same manner as in example 1.
Example 6
The present embodiment provides a method for 3D culture of laryngeal cancer tissue, comprising the steps of:
step one, taking laryngeal cancer tissues, cutting the tissues on ice, adding 5ml of mixed digestive juice I of collagenase III + cell dispersing enzyme II for heavy suspension, transferring the mixture to a shaker at 37 ℃, digesting the mixture for 150min at 220rpm, and centrifuging the mixture at 1500rpm to remove the digestive juice I. The final concentrations of collagenase III and cell dispersing enzyme II in the digestion solution I were 200U/ml and 5mg/ml, respectively.
And step two, adding 5ml of hyaluronidase and Dnase I mixed digestive juice II into the system again, wherein the final concentrations of hyaluronidase and Dnase I in the digestive juice II are 100U/ml and 0.1mg/ml respectively, performing shake digestion at 37 ℃ and 220rpm for 5min, and removing the precipitate and reserving the clear liquid after the digestion is finished.
Step three, adding DMEM/F12 into the clear liquid, mixing uniformly, passing through a 100-micron screen, centrifuging at 4 ℃ and 1200rpm for 3min, and then retaining cell sediment;
step four, adding erythrocyte lysate into the cell sediment to resuspend the cells for 5min, then centrifuging at 4 ℃ and 1200rpm for 3min, removing the supernatant, adding DMEM/F12 again for resuspension, centrifuging at 4 ℃ and 1200rpm for 3min, and removing the supernatant again;
step five, counting cells, mixing the cells and matrigel according to the proportion of 30000 cells per 30 μ L, dripping into a pore plate, dripping into the center of a 6-pore plate, dripping 3 gel drops into each pore, and using the culture dish at 37 ℃ and 5% CO 2 Standing for 10min under the condition, and solidifying Marteigel. Adding 3mL of the medium of example 1 per well at 37 ℃ 5% 2 Is cultured for 14 days, during which the medium is changed every 3 to 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 procedure is the same as 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 procedure is the same as 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 procedure is the same as 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 procedure is the same as 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 passage of laryngeal carcinoma organoids
After obtaining primary laryngeal cancer organoids according to example 8, they were subcultured as follows:
1. sucking out the culture solution from the culture dish by pipette, collecting 1-2 ml TrypLE ™ Express to digest the gel drop containing organoid, placing into incubator, and digesting at 37 deg.C for 10 min;
2. adding 10 ml of sterile Hank's balanced salt solution, centrifuging at 1000 rpm for 3min;
3. discarding the supernatant, adding 200. Mu.l of the medium of example 1 to resuspend the organoid, adding 250. Mu.l of Matrigel, mixing, dropping to a new 35 mm petri dish, standing for 5min, transferring into an incubator, inverting, after 40 min, supplementing 2-4 ml of the medium of example 3, and standing and culturing at 37 ℃.
Subculturing for 10 days to obtain second generation laryngocarcinoma organoid, subculturing for 45 days to obtain 6 th generation laryngocarcinoma organoid, and obtaining laryngocarcinoma organoid contrast diagram after 6 passages as shown in FIG. 6, which has good organoid structure and can maintain morphological structure of original tissue after 6 generations.
Example 12
Cryopreservation resuscitation of laryngocarcinoma organoids
After obtaining primary laryngeal cancer organoids according to example 8, they were cryopreserved as follows:
sucking out the culture solution from the culture dish by using a pipettor, digesting the gel drops containing the organoids by taking 1-2 ml of cell recovery solution at room temperature for 5-10 min, repeatedly blowing by using the pipettor, and blowing off the gel drops; adding 10 ml of sterile Hank's balanced salt solution, centrifuging at 1000 rpm for 3min; the supernatant was discarded, and 1ml of a cryopreservation solution (90% FBS +10% DMSO) was added to resuspend the organoids, transferred to a cryopreservation tube, cooled down by a program (4 ℃ → -20 ℃ → -80 ℃ → liquid nitrogen), and finally frozen in liquid nitrogen. Or freezing and storing in a refrigerator at-80 deg.C.
The resuscitation operation of the laryngeal cancer organoids after cryopreservation is as follows:
(1) Taking out the frozen tube, directly immersing the frozen tube in warm water at 37 ℃, and shaking the frozen tube to melt the frozen tube as soon as possible without delay.
(2) And (3) taking out the cryopreserved pipe from the water bath at 37 ℃, moving the cryopreserved pipe to a biological safety cabinet, opening a cover, sucking out cell suspension by using a suction pipe, adding the cell suspension into a centrifuge tube, dropwise adding more than 10 times of SAGM culture medium, and uniformly mixing. 1000 Centrifuge at rpm for 3 min.
(3) The supernatant was discarded, 120. Mu.l of the medium of example 3 was added to resuspend the organoid, 150. Mu.l of Matrigel was added thereto, mixed well, dropped onto a 35 mm petri dish, allowed to stand for 5min, transferred into an incubator, inverted, and after 40 min, 2 to 4 ml of the medium of example 3 was supplemented, and allowed to stand for culture at 37 ℃.
(4) The culture medium was changed the next day, and laryngeal cancer organoids were obtained after further culturing for 4 days as shown in FIG. 7.
Comparative example 1
The present comparative example provides a method for culturing laryngeal cancer organoids by directly using the existing culture medium commonly used for organoid culture, comprising the following steps:
the procedure was carried out in the same manner as in example 6 using a normal medium (DMEM +10% FBS), and as a result, the laryngeal cancer cells were adhered to the bottom of the culture dish during the culture, and similar to the normal cell culture results, no structural, multicellular organoids could be formed. FIG. 8 provides a structural diagram of organoid morphology at 14 days of culture in this comparative example.
Comparative example 2
This comparative example provides a laryngeal cancer culture medium, which is different from example 1 in that EGF is not added, and the method for culturing laryngeal cancer organoids using the laryngeal cancer culture medium is the same as example 6. As a result, the formation of the throat cancer organoid was extremely slow, small in number and small in diameter, and FIG. 9 provides a structural diagram of organoid morphology at 14 days of culture in this comparative example.
Comparative example 3
This comparative example provides a laryngeal cancer culture medium, which is different from example 1 in that R-spondin 1 is not added, and the method for culturing laryngeal cancer organoids using the laryngeal cancer culture medium is the same as example 6. As a result, the formation of throat cancer organoids was slow and the growth rate was slow, and FIG. 10 provides a structural drawing of organoid morphology for 14 days of culture in this comparative example.
In conclusion, organoids formed by the laryngeal cancer tissue organoid culture medium can maintain the tissue cell specificity, stem cell characteristics and genotyping highly consistent, and the cell morphology and physiological functions are also highly similar. The culture medium can finish cryopreservation, resuscitation and passage of the laryngeal cancer organoid, meets the requirement of large-scale replication of the laryngeal cancer organoid, and controls the laryngeal cancer organoid obtained by culture to have high consistency.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to 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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