CN114774359B - Cervical squamous carcinoma organoid culture medium and construction method thereof - Google Patents
Cervical squamous carcinoma organoid culture medium and construction method thereof Download PDFInfo
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- CN114774359B CN114774359B CN202210298185.3A CN202210298185A CN114774359B CN 114774359 B CN114774359 B CN 114774359B CN 202210298185 A CN202210298185 A CN 202210298185A CN 114774359 B CN114774359 B CN 114774359B
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Abstract
The invention relates to the technical field of organoid culture, and particularly discloses a cervical squamous carcinoma organoid culture medium and a construction method thereof. The cervical squamous carcinoma organoid culture medium comprises the following components: a basal culture medium, an HEPES buffer pair, a penicillin streptomycin solution, a nutritional additive and a specific factor; the specific factors comprise nicotinamide, N-acetylcysteine, Y-27632, noggin factor, A83-01, EGF, FGF10, forskolin and hydrocortisone. The invention develops the cervical squamous carcinoma organoid culture medium aiming at cervical squamous carcinoma, improves the culture success rate and long-term maintenance rate of the cervical carcinoma organoid, and the cultured cervical squamous carcinoma organoid can maintain the histological morphology and genetic characteristics of original tumor tissues; the developed cervical squamous carcinoma organoid culture medium is beneficial to providing a new idea for the research of cervical carcinoma.
Description
Technical Field
The invention relates to the technical field of organoid culture, in particular to a cervical squamous carcinoma organoid culture medium and a construction method thereof.
Background
Cervical cancer is one of the most common gynecological tumors. 2021 global statistical analysis of tumors suggested that the incidence and mortality of cervical cancer were the fourth of all gynecological tumors. The most common types of pathology for cervical cancer are squamous cell carcinoma and adenocarcinoma, accounting for 70% and 25% of all cases, respectively. Squamous carcinomas originate in cervical squamous epithelia and adenocarcinomas in columnar epithelia. To date, cervical cancer research has relied on a limited number of cell lines, xenografts, and transgenic mouse models. Widely used cell lines such as hela, siha have been partially deprived of generalisation of clinical patient characteristics and have limited preclinical research value through long-term in vitro culture selection. However, the xenograft and transgenic models cannot be widely applied in research due to high price, long culture period and the like. Therefore, we established an economical and simple model representative of patients for the study of cervical cancer.
Cancer research relies on a stable supply of cells-normal and cancerous cells that can be cultured in the laboratory. The tumor organoid is an in vitro 3D culture model developed in recent years, the model simulates extracellular matrix by using materials such as matrigel or hydrogel and the like in vitro, provides a three-dimensional bracket for cells, exogenously adds substances such as small molecules, growth factors and the like to form a stem cell ecological environment, induces self-renewal and differentiation of tissues or stem cells to form a three-dimensional cell structure, and the organoid model is closely related to source tissues and reproduces the cell structure and genetic traits of the source tissues. The genetic heterogeneity and biological characteristics of the patient tumor can be well reproduced in vitro by constructing the tumor organoids derived from the patient, and the patient individualized treatment scheme can be guided by performing high-throughput drug screening on the organoids by combining next generation sequencing. In the research of tumor mechanism, organoid model can be used as supplement between cell experiment and in vivo experiment, and organoid from patient can be used as bridge between animal model and human clinical experiment.
However, the existing culture success rate for culturing the cervical squamous cell carcinoma organoid in the prior art is low, and only has a success rate of 50%, so that a cervical squamous cell carcinoma organoid culture medium with high culture success rate is needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a cervical squamous carcinoma organoid culture medium and a construction method thereof. The cervical squamous carcinoma organoid culture medium developed aiming at cervical squamous carcinoma can improve the success rate and long-term maintenance rate of cervical carcinoma organoid culture, and is favorable for providing a new idea for cervical carcinoma research.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a cervical squamous carcinoma organoid culture medium, which comprises the following components:
a basal culture medium, a HEPES buffer pair, a penicillin streptomycin solution, a nutritional additive and a specific factor;
the specific factor is nicotinamide, N-acetylcysteine, Y-27632, noggin factor, A83-01, EGF, FGF10, forskolin and hydrocortisone.
Through a large amount of researches and experiments, the inventor of the invention discovers that a culture medium for the cervical squamous carcinoma organoid is developed, the culture success rate and the long-term maintenance rate of the cervical squamous carcinoma organoid are improved, the culture success rate of the cervical squamous carcinoma organoid is improved from 50% to 73%, the biological activity of the cervical squamous carcinoma organoid is well maintained, the growth activity and the morphology of the organoid are not influenced, and the cultured cervical squamous carcinoma organoid can maintain the histological morphology and the genetic characteristics of the original tumor tissue.
Wherein, the addition of HEPES buffer to the culture medium can promote the biological activity of organoid and prevent the adverse effect of pH fluctuation of the culture medium on the cell growth. The specific factors can be added to synergistically improve the culture success rate of cervical squamous carcinoma organoids.
As a preferred embodiment of the cervical squamous carcinoma organoid culture medium of the invention, the cervical squamous carcinoma organoid culture medium comprises the following components:
basic culture medium, HEPES buffer pair 10-12 mM, penicillin streptomycin solution 1-2%, nutrient additive 1-3%, nicotinamide 10-12 mmol/L, N-acetylcysteine 1-1.5 mmol/L, Y-27632 2-10 mu M, noggin factor 80-120 ng/ml, A83-01-450 nmol/L, EGF-10 ng/ml, FGF 10-120 ng/ml, forskolin 8-12 mu M and hydrocortisone 400-600 ng/ml.
As a preferred embodiment of the cervical squamous carcinoma organoid culture medium of the invention, the cervical squamous carcinoma organoid culture medium comprises the following components:
basal medium, HEPES buffer pair 10mM, penicillin streptomycin solution 1%, nutritional supplement 3%, nicotinamide 10mmol/L, N-acetylcysteine 1.25mmol/L, Y-27632 10. Mu. M, noggin factor 100ng/ml, A83-01 500nmol/L, EGF ng/ml, FGF10 100ng/ml, forskolin 10. Mu.M and hydrocortisone 500ng/ml.
As a preferred embodiment of the cervical squamous carcinoma organoid culture medium, the basic culture medium is advanced DMEM/F12; the nutritional additive is at least one of Glutamax, B27 and N2. More preferably, the nutritional additive is a combination of Glutamax, B27 and N2. The addition of the nutrient is beneficial to the culture of the cervical squamous carcinoma organoid, so that the growth form of the cervical squamous carcinoma organoid is better.
As a preferred embodiment of the cervical squamous carcinoma organoid culture medium, the volume ratio of the nutritional additive to the specific factor is 10: (0.5-1.5). More preferably, the volume ratio of the nutritional supplement to the specific factor is 10:0.8.
when the nutrient additive and the specific factor are compounded in a specific volume ratio, the culture effect of the cervical squamous carcinoma organoid can be improved.
The invention provides a method for constructing a cervical squamous carcinoma organoid model, which comprises the following steps:
1) Obtaining cervical cancer tissues, mechanically and enzymatically digesting the cervical cancer tissues, filtering and centrifuging, retaining cell precipitates, and removing red blood cells to obtain cell masses;
2) Resuspending the cell pellet in matrigel, inoculating, and fixing; culturing in the above-mentioned cervical squamous carcinoma organoid culture medium, observing and changing liquid every 2-3 days, and subculturing every 7-21 days.
As a preferred embodiment of the method for constructing the organoid model of cervical squamous carcinoma of the present invention, in step 1), the cervical cancer tissue is digested with collagenase II at a concentration of 0.5 mg/ml.
As a preferred embodiment of the method for constructing the organoid model of cervical squamous carcinoma of the present invention, in step 2), the cell mass is resuspended in matrigel at a density of 1 ten thousand cells/10. Mu.l.
In a third object, the invention provides the use of the culture medium in culturing cervical squamous carcinoma organoids.
Compared with the prior art, the invention has the following beneficial effects:
the invention develops the cervical squamous carcinoma organoid culture medium aiming at cervical squamous carcinoma, improves the culture success rate and the maintenance rate after passage of the cervical carcinoma organoid, and the cultured cervical squamous carcinoma organoid can maintain the histological morphology and the genetic characteristics of the original tumor tissue; the developed cervical squamous carcinoma organoid culture medium is beneficial to providing a new idea for the research of cervical carcinoma.
Drawings
FIG. 1 is a light microscopic image of a cervical squamous carcinoma organoid obtained after culture in the cervical squamous carcinoma organoid culture medium of example 1;
FIG. 2 is a light microscopic image of a cervical squamous carcinoma organoid obtained after culture in the cervical squamous carcinoma organoid culture medium of example 2;
FIG. 3 is a comparative statistical plot of the effect of the cervical squamous carcinoma organoid culture media of example 2 and comparative examples 1-12 on cervical squamous carcinoma organoid size;
fig. 4 is an optical microscope photograph of cervical squamous carcinoma organoids obtained after culture in the cervical squamous carcinoma organoid culture medium of comparative example 13.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples and comparative examples, the experimental methods used were all conventional ones unless otherwise specified, and the materials, reagents and the like used were commercially available.
In the following examples and comparative examples, the nutritional additives include Glutamax, B27 and N2; the specific factor is nicotinamide, N-acetylcysteine, Y-27632, noggin factor, A83-01, EGF, FGF10, forskolin and hydrocortisone.
In the following examples and comparative examples, penicillin streptomycin solution (100 Xpenicillin streptomycin mixed solution), hepes buffer pair, glutamax, B27, N2 were purchased from England Weiji (Shanghai) trade Co., ltd.; nicotinamide (nicotinamide), N-acetylcysteine (N-acetylcysteine), forskolin, A83-01, available from Sigma-Aldrich; noggin factor, EGF, FGF10, R-spondin 1, CHIR99021 from PeproTech; y-27632, hydrocortisone (Hydrocortisone) was purchased from stem cell.
Example 1 cervical squamous carcinoma organoid culture Medium
This example provides a cervical squamous carcinoma organoid culture medium comprising the basal medium Advanced DMEM/F12, 12mM Hepes buffer pair, 2% penicillin streptomycin solution, 1% Glutamax, 1% B27, 1% N2, 12mmol/L nicotinamide (nicotinamine), 1.25mmol/L N-acetylcysteine (N-acetylcysteine), 2 μ M Y-27632, 100ng/ml Noggin factor, 500nmol/L A-83-01, 10ng/ml EGF, 100ng/ml FGF10, 10 μ M Forskolin, and 500ng/ml hydrocortisone. The concentration of each component of the specific factor is based on the concentration of the specific factor in the culture medium of the cervical squamous carcinoma organoid.
Wherein, the volume ratio of the nutritional additive to the specific factor is 10:0.8.
example 2 cervical squamous carcinoma organoid culture Medium
This example provides a cervical squamous carcinoma organoid medium comprising the basal medium Advanced DMEM/F12, 10mM Hepes buffer pair, 1% penicillin streptomycin solution, 1% Glutamax, 1% B27, 1% N2, 10mmol/L nicotinamide (nicotinamine), 1.25mmol/L N-acetylcysteine (N-acetylcysteine), 10 μ M Y-27632, 100ng/ml Noggin factor, 500nmol/L A-01, 5ng/ml EGF, 100ng/ml FGF10, 10 μ M Forskolin, and 500ng/ml hydrocortisone. The concentration of each component of the specific factor is based on the concentration of the specific factor in the culture medium of the cervical squamous carcinoma organoid.
Wherein, the volume ratio of the nutritional additive to the specific factor is 10:0.5.
example 3 cervical squamous carcinoma organoid culture Medium
This example provides a cervical squamous carcinoma organoid culture medium comprising the basal medium Advanced DMEM/F12, 10mM Hepes buffer pair, 1% penicillin streptomycin solution, 0.3% Glutamax, 0.3% B27, 0.4% N2, 10mmol/L nicotinamide (nicotinamine), 1.0mmol/L N-acetylcysteine (N-acetylcysteine), 5 μ M Y-27632, 80ng/ml Noggin factor, 450nmol/L A-01, 5ng/ml EGF, 80ng/ml FGF10, 8 μ M skForolin, and 400ng/ml hydrocortisone. The concentration of each component of the specific factor is based on the concentration of the specific factor in the culture medium of the cervical squamous carcinoma organoid.
Wherein, the volume ratio of the nutritional additive to the specific factor is 10:1.5.
example 4 cervical squamous carcinoma organoid culture Medium
This example provides a cervical squamous carcinoma organoid medium comprising the basal medium Advanced DMEM/F12, 10mM Hepes buffer pair, 1% penicillin streptomycin solution, 1% Glutamax, 1% B27, 1% N2, 10mmol/L nicotinamide (nicotinamine), 1.5mmol/L N-acetylcysteine (N-acetylcysteine), 10 μ M Y-27632, 120ng/ml Noggin factor, 550nmol/L A-83-01, 10ng/ml EGF, 120ng/ml FGF10, 12 μ M Forskolin, and 600ng/ml hydrocortisone. The concentration of each component of the specific factor is based on the concentration of the specific factor in the culture medium of the cervical squamous carcinoma organoid.
Wherein, the volume ratio of the nutritional additive to the specific factor is 10:0.8.
A construction method of a cervical squamous carcinoma organoid model comprises the following steps:
1) Sample pretreatment: washing the cervical cancer tissue specimen obtained by resection/biopsy for 3-5 times by using normal saline to remove surface impurities, filling the cervical cancer tissue specimen into an Advanced DMEM/F12 culture medium containing 2% of double-antibody amphotericin B for preservation, and transporting the cervical cancer tissue specimen on ice;
2) Sample digestion: in a biosafety cabinet, the samples were transferred to 10cm sterile petri dishes and 1ml of 0.5mg/ml collagenase II (digest, substrate: advanced DMEM/F12 medium with 2% diabase and amphotericin B) was added and the tissue was minced to 1-5mm with sterile ophthalmic scissors 3 Transferring the tissue blocks and the digestive juice into a 50ml centrifuge tube, supplementing the digestive juice according to a proper amount of tissue volume, and carrying out shake digestion at 37 ℃ and 120rpm for 120mins;
3) Sample dissociation: terminating digestion with PBS of the same volume, filtering the digested tissue suspension with a cell sieve of 100 μm, collecting the unfiltered tissue fragments on the cell sieve, placing the unfiltered tissue fragments in a 50ml centrifuge tube, adding 3ml of tryple (plus or minus according to the number of fragments), digesting for 10mins at 37 ℃ and 120rpm by shaking;
4) Cell filtration: terminating digestion by PBS with the same volume, filtering the digested tissue suspension by a 100-micron cell sieve again, collecting filtrate obtained by filtering twice, centrifuging for 5min at 4-600 g, and retaining cell precipitate to remove supernatant;
5) Lysed erythrocytes (optional): mixing red blood cells in the visible cell precipitate, adding 1-2ml of red blood cell lysate, resuspending the cells, and lysing for 2min at room temperature;
6) Cell washing: terminating the lysis by using 10ml of PBS, centrifuging for 5min at 4-600 g, retaining cell sediment, removing supernatant, and repeating twice;
7) Inoculation: re-suspending the cell precipitate with organoid culture medium and matrigel (1:2 in volume ratio) at a density of 1 ten thousand cells/10 mul, inoculating 30 mul/well into a 48-well plate, carefully inverting the plate, fixing for 30mins in a 37-degree incubator, and adding 200ul culture medium to cover the gel drop after the gel is solidified;
8) Culturing: culturing in the above-described cervical squamous carcinoma organoid medium of examples 1-4 at 37 ℃ and 5% CO 2 The concentration is observed every 2-3 days, and the liquid is changed every 7-21 days for passage.
Cervical squamous carcinoma organoids obtained after culturing in the cervical squamous carcinoma organoid culture medium of example 1-2 above were observed under an inverted light microscope according to the construction method of example 5, as shown in fig. 1-2. The morphology of the cervical squamous carcinoma organoid obtained after the culture in the cervical squamous carcinoma organoid culture medium of examples 3-4 under an inverted light microscope is similar to that of the cervical squamous carcinoma organoid shown in FIGS. 1-2, and therefore, the description thereof is omitted.
Comparative examples 1 to 12
On the basis of example 2, comparative examples 1 to 12 show that the culture medium for the cervical squamous cell carcinoma organoid is prepared by subtracting or adding the specific factors shown in Table 1, and then the cervical squamous cell carcinoma organoid is cultured for 7 days according to the construction method of the cervical squamous cell carcinoma organoid model shown in example 5, and then 5 visual fields are randomly selected under an inverted microscope for measuring the diameter of the organoid.
TABLE 1
The effect of the cervical squamous carcinoma organoid medium of comparative examples 1-12 on cervical squamous carcinoma organoid formation is shown in FIG. 3, wherein "-" in FIG. 3 represents the medium of comparative example 2 minus the corresponding factor and "+" represents the medium of comparative example 2 plus the corresponding factor.
The results show that the diameters of the cervical squamous carcinoma organoids cultured by the cervical squamous carcinoma organoid culture medium of the comparative examples 1-12 are less than the culture effect of the cervical squamous carcinoma organoid culture medium of the example 2, wherein one of Noggin, A83-01, EGF, FGF7, FGF10, N-acetylcysteine, nicotinamide, hydrocortisone, forskolin and Y-27632 is removed from the comparative examples 1-3 and 5-10 respectively, and the culture effect of the cervical squamous carcinoma organoids is less than that of the example 2, which shows that the Noggin, A83-01, EGF, FGF7, FGF10, N-acetylcysteine, nicotinamide, hydrocortisone, forskolin and Y-27632 cooperate with each other to promote the culture effect of the cervical squamous carcinoma organoids and improve the success rate of culturing the cervical carcinoma organoids. The culture medium of comparative example 4 is added with FGF7 factor, the culture medium of comparative example 11 is added with R-spondin 1, the culture medium of comparative example 12 is added with R-spondin 1 and CHIR99021, the sizes of the cervical squamous cell carcinoma organoids after being cultured by the three culture media are smaller than those of example 2, and the technical effect of the invention can be obtained without adding any specific factor.
At the same time, the maintenance rate after passage of organoids in the culture medium of each example (the organoids are digested again into single cell suspension and planted in matrigel, the successful passage is defined when the single cells grow into organoids again, and the long-term maintenance is defined as the organoids are cultured for more than 10 generations) is observed, and all the comparative examples except examples 1 to 4 and comparative example 12 can not pass through 3 generations.
The inventor finally obtains the cervical squamous carcinoma organoid culture medium through a large number of experimental verifications, which comprises the following components: 10-12 mM of basal culture medium, 10-12 mM of Hepes buffer pair, 1-2% of penicillin streptomycin solution, 1-3% of nutritional additive, 10-12 mmol/L, N-acetylcysteine 1-1.5 mmol/L, Y-27632 0-10 mu M, noggin factor 80-120 ng/ml, A83-01-450 nmol/L, EGF-10 ng/ml, FGF 10-120 ng/ml, forskolin 8-12 mu M and hydrocortisone 400-600 ng/ml.
Comparative example 13
Compared with example 2, the volume ratio of the nutritional supplement to the specific factor is 10:3, the remaining components and concentrations were the same as in example 2, with reference to fig. 4.
Test example, effect of Cervix squamous cell carcinoma organoid culture Medium on Cervix squamous cell carcinoma culture success ratio
The groups (examples 1-4 and comparative examples 11-13) of the experimental example incorporate 60 cervical squamous carcinoma organoid samples, and the activity of the cervical squamous carcinoma organoids obtained by the culture of the cervical squamous carcinoma organoid culture medium of the above examples 1-4 and comparative examples 11-13 is detected according to the construction method of example 5, and the success rate of culturing the cervical carcinoma organoids of each group is shown in table 2, wherein successful culture means that the tumor cells or cell clusters digested under specific culture conditions can form a three-dimensional structure in vitro, i.e. organoid culture is successful.
TABLE 2
| Group of | Success rate of cervical cancer organoid culture (%) |
| Example 1 | 67％ |
| Example 2 | 73％ |
| Example 3 | 63％ |
| Example 4 | 58％ |
From the above results, it can be seen that 44 cases of cervical squamous cell carcinoma organoids obtained by culturing with the cervical squamous cell carcinoma organoid medium of example 2 can be successfully obtained, the success rate can be increased to 73% (44/60), and the effect of examples 1, 3-4 on successfully culturing cervical squamous cell carcinoma organoids is similar to that of example 2.
The culture medium of comparative examples 11-12 is newly added with other specific factors, which have a lower effect of culturing the cervical squamous carcinoma organoids than that of example 2, and shows that the specific factors are not added randomly, and the addition of some specific factors can influence the organoid culturing effect.
The culture medium of comparative example 13 changes the volume ratio of the nutrient additive and the specific factor, the success rate of culturing the cervical squamous cell carcinoma organoid is lower than that of example 2, and the fact that the nutrient additive and the specific factor are matched in a specific volume ratio can improve the success rate of culturing the cervical squamous cell carcinoma organoid.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (7)
1. The cervical squamous carcinoma organoid culture medium is characterized by comprising the following components:
a basal culture medium, a HEPES buffer pair of 10-12 mM, a penicillin streptomycin solution of 1-2%, a nutritional additive of 1-3%, nicotinamide of 10-12 mmol/L, N-acetylcysteine of 1-1.5 mmol/L, Y-27632 of 2-10 mu. M, noggin factor of 80-120 ng/ml, A83-01-450 nmol/L, EGF of 5-10 ng/ml, FGF of 10-120 ng/ml, forskolin of 8-12 mu M and hydrocortisone of 400-600 ng/ml; the nutritional additive is at least one of Glutamax, B27 and N2; nicotinamide, N-acetylcysteine, Y-27632, noggin factor, A83-01, EGF, FGF10, forskolin and hydrocortisone are used as specific factors;
the volume ratio of the nutritional additive to the specific factor is 10: (0.5-1.5).
2. The cervical squamous carcinoma organoid medium of claim 1, consisting of:
basal medium, HEPES buffered pair 10mM, penicillin streptomycin solution 1%, nutritional supplement 3%, nicotinamide 10mmol/L, N-acetylcysteine 1.25mmol/L, Y-27632 10 μ M, noggin factor 100ng/ml, A83-01 nmol/L, EGF 5ng/ml, FGF10 100ng/ml, forskolin10 μ M and hydrocortisone 500ng/ml.
3. The cervical squamous carcinoma organoid medium of claim 1, wherein said basal medium is advanced DMEM/F12.
4. A construction method of a cervical squamous carcinoma organoid model is characterized by comprising the following steps:
1) Obtaining cervical cancer tissues, mechanically and enzymatically digesting the cervical cancer tissues, filtering and centrifuging, retaining cell precipitates, and removing red blood cells to obtain cell masses;
2) Resuspending the cell pellet in matrigel, inoculating, and fixing; culturing in the cervical squamous carcinoma organoid culture medium of any of claims 1 to 3, and carrying out subculture every 7 to 21 days with fluid change observed every 2 to 3 days.
5. The method for constructing the organoid model of cervical squamous carcinoma according to claim 4, wherein in step 1), the cervical carcinoma tissue is digested with collagenase II at a concentration of 0.5 mg/ml.
6. The method of claim 4, wherein in step 2), the cell mass is resuspended in matrigel at a density of 1 ten thousand cells/10 μ l.
7. Use of the cervical squamous carcinoma organoid medium of any of claims 1 to 3 for culturing a cervical squamous carcinoma organoid.
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