CN117230011A - Construction method of ovarian boundary tumor organoid model - Google Patents
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Abstract
The invention relates to the field of biochemistry, and particularly discloses a construction method of an ovarian juncture tumor organoid model; according to the invention, a plurality of models are built under an environment of consistent and stable in vitro by collecting fresh tumor tissues of a plurality of patients, and the models are subjected to cystic expansion, stabilization or mixing, each expansion type can be kept stable for a long time, meanwhile, the in vitro culture time of the models is more than three months, the maximum in vitro expansion time of the PDO models is more than eight months, the models have no obvious morphological change or slower expansion speed, the PDO models created by the invention can summarize the markers of the primary tissues, and the diagnostic molecular markers of BOT (BOT) such as ER, P53, pan-CK, PAX8 and Ki67 are well expressed in the primary tumors and the organoids, so that experimenters can carry out treatment experiments by taking the organoids as a platform, and the accuracy of experimental results is effectively ensured.
Description
Technical Field
The invention belongs to the technical field of biochemistry, in particular to a construction method of an ovarian juncture tumor organoid model,
background
The Boundary Ovarian Tumor (BOT) is a special class of tumor that is intermediate between benign and malignant ovaries, is insensitive to traditional chemotherapy regimens, and has limited development of targeted drugs due to the lack of representative cell lines.
Organoid culture techniques allow single cells, typically derived from stem cells, to grow into small clusters in an in vitro three-dimensional (3D) environment in which dispersed cells self-assemble and differentiate into functional cell clusters. Compared with the traditional two-dimensional model, the tumor organoids can better reproduce cytogenetic characteristics, structures and functions of primary tissues. Therefore, the organoid model is widely applied to transformation application researches such as anti-tumor drug discovery, personalized drug development and the like. It should be noted that tumor cells are subjected to extreme replicative stresses and are susceptible to genomic damage, often resulting in DNA Double Strand Breaks (DSBs), whereas tumor cell repair DSBs often use homologous recombination repair or non-homologous end joining repair pathways. In theory, the repair of DSB in tumor cells can be effectively inhibited by using homologous recombination or non-homologous end joining inhibitors, thereby achieving the effect of inhibiting tumor growth.
Disclosure of Invention
The invention aims to provide a construction method of an ovarian boundary tumor organoid model, which aims to solve the problems in the background technology,
in order to achieve the above purpose, the present invention provides the following technical solutions:
the construction method of the ovarian boundary tumor organoid model specifically comprises the following steps:
s1, cutting a fresh ovarian boundary tumor tissue sample into fragments of 1-2 mm, flushing blood with ice-cold PBS, and removing fat and necrotic tissues as much as possible; collecting proper tissue embedded frozen section for pathological staining, digesting the rest sample with dispersive enzyme ll (1U/ml) at 37 ℃ for about 30 minutes, taking a small amount of supernatant to observe the size of the cell mass, preferably 10-20 cell mass every 1-2 minutes, adding 20 times of volume of washing liquid to dilute the digestive enzyme after digestion is finished, standing for 3 minutes at room temperature to precipitate large undigested tissue, collecting the culture solution containing the cell mass, continuously adding dispersive enzyme II after collecting the digestive solution, continuously digesting the tissue at 37 ℃ to obtain more cell mass, filtering the culture solution with a 70 mu M cell sieve, flushing the cell mass on the cell sieve with 1640 cell culture solution to discard single cells such as red blood cells, obtaining initial organoid glands, collecting the primary organoid glands, embedding the organoid glands in 70% matrigel, inoculating in a 48-degree pore plate, placing in a 37-degree incubator to solidify matrigel, adding growth medium for continuous culture, replacing the culture medium with fresh culture medium every 3 days, and carrying out passage every 2-3 weeks;
s2, replacing liquid and passaging of the organoid, discarding the organoid culture liquid in a 48-pore plate, carefully adding 500 μl of PBS liquid from the edge of the 48-pore plate to wash the culture hole, after discarding the washing liquid, adding 300 μl of fresh culture liquid to continue culturing, passaging when the confluence of the organoid reaches more than 70%, discarding the culture liquid during passaging, adding 200 μl of washing liquid, using a P1000 gun head to break matrigel containing the organoid, blowing matrigel and organoid for multiple times, blowing and breaking the organoid to 20 cell mass, collecting organoid liquid, centrifuging to obtain organoid fragments, adding 70% matrigel, planting the organoid in the 48-pore plate, placing in a 37-degree incubator to solidify the matrigel, and adding the culture liquid to continue culturing;
s3, washing the organoids with ice-cold PBS, adding a cell recovery liquid to digest matrigel, centrifugally separating the organoids, fixing the organoids with 4% paraformaldehyde, dehydrating the organoids in a 30% sucrose solution for 24 hours, embedding the organoids in 0.C.T ice gel for slicing, treating the slices with dimethylbenzene and gradient ethanol, repairing antigens with a citric acid buffer solution, permeabilizing and blocking the organoids, adding a corresponding primary antibody and a fluorescent-labeled secondary antibody, staining cell nuclei, fixing the glass slide with neutral resin, and photographing and observing under a fluorescent microscope;
s4, after culturing for 48 hours in a 48-well plate, adding 1-50 mu M of Bractoppin into the organoid for two days, adding 20ul of XTT/PMS solution into the organoid culture medium for further culturing for 2 hours, evaluating the activity of the organoid by using a microplate reader, calculating the semi-inhibition concentration values of several organoids, staining the organoid by using a cell 3D live-dead disease detection kit, marking the live cells and the dead cells as green and red respectively, and photographing by using a fluorescence microscope;
s5, dissolving the Bractoppin in sterile water to obtain 10mM stock solution, subpackaging, storing the solution at-80 degrees, and diluting to corresponding working concentration when in use;
s6, completely lysing protein extraction and western blot cells in RIPA protein lysis buffer, centrifuging to obtain protein solution, measuring protein concentration, adding LDS buffer, heating for denaturation, separating protein by using SDS-PAGE gel, transferring to a membrane, sealing, incubating with primary antibody and secondary antibody, developing with ECL luminescent solution, and photographing;
s7, culturing cells and analyzing the cells of H8910 and SKOV3 in 10% fetal bovine serum, changing a culture medium every 3 days, routinely checking mycoplasma pollution, determining sensitivity of the cells to medicines by using a CCK8 kit, adding 10ml of a CCK8 reagent after the cells grow to 60% in a 96-well plate, and detecting proliferation activity of the cells by using an enzyme-labeled instrument after culturing for 2 hours;
s8, analyzing the influence of the brachypin on cells by using a cell cycle and apoptosis detection kit (C1052, beyotime), collecting the cells by using a scraper, flushing the cells by using PBS after 24 hours of drug treatment, adding the cells to 75% ethanol overnight when carrying out cell cycle analysis, re-suspending the cells into a single cell solution, marking the single cell solution by using PI, then carrying out cell cycle research by using flow cytometry, staining and detecting the apoptosis by using 5 mu l of annexin V-FITC and 10 mu l of PI staining solution, and carrying out flow cytometry after incubating in darkness for 20 minutes;
s9, performing experiments by using a comet detection kit according to the specification, collecting cells by using a rubber scraper after the treatment of the brachypin for 24 hours, cleaning the cells by using PBS, then re-suspending the cells in a soft agarose package and loading the cells on a glass slide coated with agarose, adding alkaline lysate into the gel to dissolve the cells after gel solidification, and performing electrophoresis for 30 minutes in an alkaline environment, and for evaluating the DNA damage level, staining the DNA by using a nucleic acid dye, photographing by using a fluorescence microscope, and determining the distance between the comet tail and the center of a cell nucleus;
s10, analyzing and detecting the functions of DNA non-homologous end connection and homologous recombination, transfecting a plasmid into cells by using a liposome 3000, inoculating the cells into a 24-well plate, replacing a culture medium after 6 hours, adding 5mM of the brachypin, discarding the culture medium, adding a fluorescein substrate, and detecting the activity of the cells by using an enzyme-labeled instrument;
s11, extracting total RNA from organoids by using an RNA extraction kit, generating a sequencing library by using an RNA library building kit, sequencing on a PE150 platform, downloading and cleaning the original data, and comparing the sequences by using HISAT2 software package and Deseq2 software.
Preferably, a plurality of the models are established, and a plurality of the models are cultured in an in vitro consistent environment.
Preferably, the organoid model requires an average incubation time in vitro of more than 3 months, and the organoid model has an in vitro amplification validation time of up to more than 8 months.
Preferably, in the step S1, the detergent composition includes: 1X 1640 cell culture solution, Y-27632 (10. Mu.M).
Preferably, in said S1, the organoid culture medium composition: 1 Xadvanced DMEM/F12 medium, A8301 (0.5. Mu.M), N-acetyl-L-cysteine (1.25 mM), nicotinamide (5 mM), estradiol (2 Mm), B27 (1X), N2 (1X), ITS (1X), glutamine (1X), HEPES (1X), EGF (50 ng/ml), FGF10 (50 ng/ml), R-spindin (200 ng/ml), noggin (100 ng/ml).
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, a plurality of models are built in an in-vitro consistent and stable environment by collecting a plurality of fresh tumor tissues from patients, and the models are in cystic expansion, solidity or mixing, and each expansion type can be kept stable for a long time. Meanwhile, the in vitro culture time of the models is more than three months, wherein the maximum in vitro amplification time of the PDO model is more than eight months, and the models have no obvious morphological change or obviously slow expansion speed. The PDO model created by the invention can reproduce BOT initial tissue molecular markers such as ER, P53, pan-CK, PAX8 and Ki67 which are well expressed in primary tumors and organoids, and experimental staff can further develop therapeutic drug experiments by taking the borderline tumor organoids as a platform.
Drawings
FIG. 1 is a graph showing the growth morphology of BOT organoids of the present invention at various time points;
FIG. 2 shows immunofluorescent staining of primary BOT tissues and organoids according to the present invention,
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Although the described embodiments are only some, not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention,
examples:
referring to fig. 1-2, the method for constructing the ovarian boundary tumor organoid model specifically comprises the following steps:
s1, cutting a fresh ovarian boundary tumor tissue sample into fragments of 1-2 mm, flushing blood with ice-cold PBS, and removing fat and necrotic tissues as much as possible; collecting proper tissue embedded frozen section for pathological staining, digesting the rest sample with dispersive enzyme ll (1U/ml) at 37 ℃ for about 30 minutes, taking a small amount of supernatant to observe the size of the cell mass, preferably 10-20 cell mass every 1-2 minutes, adding 20 times of volume of washing liquid to dilute the digestive enzyme after digestion is finished, standing for 3 minutes at room temperature to precipitate large undigested tissue, collecting the culture solution containing the cell mass, continuously adding dispersive enzyme II after collecting the digestive solution, continuously digesting the tissue at 37 ℃ to obtain more cell mass, filtering the culture solution with a 70 mu M cell sieve, flushing the cell mass on the cell sieve with 1640 cell culture solution to discard single cells such as red blood cells, obtaining initial organoid glands, collecting the primary organoid glands, embedding the organoid glands in 70% matrigel, inoculating in a 48-degree pore plate, placing in a 37-degree incubator to solidify matrigel, adding growth medium for continuous culture, replacing the culture medium with fresh culture medium every 3 days, and carrying out passage every 2-3 weeks;
s2, liquid exchange and passage of the organoids, discarding the organoid culture liquid in the 48-well plate, carefully adding 500 μl PBS liquid from the edge of the 48-well plate, and washing the culture well. After the washing liquid is abandoned, 300 mu l of fresh culture solution is added for continuous culture, when the confluence of the organoids reaches more than 70%, the culture solution is abandoned during the passage, 200 mu l of washing solution is added, the matrigel containing the organoids is scratched by using a P1000 gun head, the matrigel and the organoids are blown and crushed for many times by the gun head, the organoids are blown and crushed to 20 cell mass sizes, organoid liquid is collected and centrifuged to obtain organoid fragments, 70% matrigel is added and planted in a 48 pore plate, and after the matrigel is solidified in a 37-DEG incubator, the culture solution is added for continuous culture;
s3, washing the organoids with ice-cold PBS, adding a cell recovery liquid to digest matrigel, centrifugally separating the organoids, fixing the organoids with 4% paraformaldehyde, dehydrating the organoids in a 30% sucrose solution for 24 hours, embedding the organoids in 0.C.T ice gel for slicing, treating the slices with dimethylbenzene and gradient ethanol, repairing antigens with a citric acid buffer solution, permeabilizing and blocking the organoids, adding a corresponding primary antibody and a fluorescent-labeled secondary antibody, staining cell nuclei, fixing the glass slide with neutral resin, and photographing and observing under a fluorescent microscope;
s4, after culturing for 48 hours in a 48-well plate, adding 1-50 mu M of Bractoppin into the organoid for two days, adding 20ul of XTT/PMS solution into the organoid culture medium, continuously culturing for 2 hours, calculating semi-inhibition concentration values of several organoids after evaluating the vitality of the organoids by using a microplate reader, staining the organoids by using a cell 3D live-dead disease detection kit, marking the live cells and the dead cells as green and red respectively, and photographing by using a fluorescence microscope;
s5, dissolving the Bractoppin in sterile water to obtain 10mM stock solution, subpackaging, storing the solution at-80 degrees, and diluting to corresponding working concentration when in use;
s6, completely lysing protein extraction and western blot cells in RIPA protein lysis buffer, centrifuging to obtain protein solution, measuring protein concentration, adding LDS buffer, heating for denaturation, separating protein by using SDS-PAGE gel, transferring to a membrane, sealing, incubating with primary antibody and secondary antibody, developing with ECL luminescent solution, and photographing;
s7, culturing cells and analyzing the cells of H8910 and SKOV3 in 10% fetal bovine serum, changing a culture medium every 3 days, routinely checking mycoplasma pollution, determining sensitivity of the cells to medicines by using a CCK8 kit, adding 10ml of a CCK8 reagent after the cells grow to 60% in a 96-well plate, and detecting proliferation activity of the cells by using an enzyme-labeled instrument after culturing for 2 hours;
s8, analyzing the influence of the brachypin on cells by using a cell cycle and apoptosis detection kit (C1052, beyotime), collecting the cells by using a scraper, flushing the cells by using PBS after 24 hours of drug treatment, adding the cells into 75% ethanol overnight when carrying out cell cycle analysis, re-suspending the cells into a single cell solution, marking the single cell solution by using PI, carrying out cell cycle research by using flow cytometry, staining and detecting the apoptosis by using 5 mu l of annexin V-FITC and 10 mu l of PI staining solution, and carrying out flow cytometry after incubation in darkness for 20 minutes;
s9, adopting a comet detection kit for experiments, performing all procedures according to the specification, collecting cells by using a rubber scraper after the treatment of the comet detection kit for 24 hours, cleaning the cells by using PBS, then re-suspending the cells in a soft agarose package and loading the cells on a glass slide coated with agarose, adding alkaline lysate into the gel to dissolve the cells after gel solidification, and performing electrophoresis for 30 minutes in an alkaline environment, dyeing the DNA by using a nucleic acid dye for evaluating the DNA damage level, photographing by using a fluorescence microscope, and determining the distance between the comet tail and the center of a cell nucleus;
s10, analyzing and detecting the functions of DNA non-homologous end connection and homologous recombination, using liposome 3000 to transfect plasmids into cells, inoculating the plasmids into a 24-hole plate, replacing a culture medium after 6 hours, adding 5mM brachopypin, discarding the culture medium, adding a fluorescein substrate, and detecting the activity of the cells by using an enzyme-labeled instrument;
s11, extracting total RNA from organoids by using an RNA extraction kit, generating a sequencing library by using an RNA library building kit, sequencing on a PE150 platform, downloading and cleaning the original data, and comparing the sequences by using HISAT2 software package and Deseq2 software. .
In order to ensure the test accuracy of the model samples, a plurality of models are built, and the models are cultured in an in-vitro consistent environment, so that the accuracy of the models is accurately measured.
In S1, the detergent composition includes: 1X 1640 cell culture solution, Y-27632 (10. Mu.M).
In the S1, organoid culture fluid composition: 1 Xadvanced DMEM/F12 medium, A8301 (0.5. Mu.M), N-acetyl-L-cysteine (1.25 mM), nicotinamide (5 mM), estradiol (2 Mm), B27 (1X), N2 (1X), ITS (1X), glutamine (1X), HEPES (1X), EGF (50 ng/ml), FGF10 (50 ng/ml), R-spindin (200 ng/ml), noggin (100 ng/ml).
The average culture time of the organoid model in vitro is more than 3 months, so as to verify the long-time stability of the organoid model, and the in-vitro amplification verification time of the organoid model is up to 8 months,
although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. The construction method of the ovarian boundary tumor organoid model is characterized by comprising the following steps of:
s1, cutting a fresh ovarian boundary tumor tissue sample into fragments of 1-2 mm, flushing blood with ice-cold PBS, and removing fat and necrotic tissues as much as possible; collecting proper tissue embedded frozen section for pathological staining, digesting the rest sample with dispersive enzyme ll (1U/ml) at 37 ℃ for about 30 minutes, taking a small amount of supernatant to observe the size of the cell mass, preferably 10-20 cell mass every 1-2 minutes, adding 20 times of volume of washing liquid to dilute the digestive enzyme after digestion is finished, standing for 3 minutes at room temperature to precipitate large undigested tissue, collecting the culture solution containing the cell mass, continuously adding dispersive enzyme II after collecting the digestive solution, continuously digesting the tissue at 37 ℃ to obtain more cell mass, filtering the culture solution with a 70 mu M cell sieve, flushing the cell mass on the cell sieve with 1640 cell culture solution to discard single cells such as red blood cells, obtaining initial organoid glands, collecting the primary organoid glands, embedding the organoid glands in 70% matrigel, inoculating in a 48-degree pore plate, placing in a 37-degree incubator to solidify matrigel, adding growth medium for continuous culture, replacing the culture medium with fresh culture medium every 3 days, and carrying out passage every 2-3 weeks;
s2, replacing liquid and passaging of the organoid, discarding the organoid culture liquid in a 48-pore plate, carefully adding 500 μl of PBS liquid from the edge of the 48-pore plate to wash the culture hole, after discarding the washing liquid, adding 300 μl of fresh culture liquid to continue culturing, passaging when the confluence of the organoid reaches more than 70%, discarding the culture liquid during passaging, adding 200 μl of washing liquid, using a P1000 gun head to break matrigel containing the organoid, blowing matrigel and organoid for multiple times, blowing and breaking the organoid to 20 cell mass, collecting organoid liquid, centrifuging to obtain organoid fragments, adding 70% matrigel, planting the organoid in the 48-pore plate, placing in a 37-degree incubator to solidify the matrigel, and adding the culture liquid to continue culturing;
s3, washing the organoids with ice-cold PBS, adding a cell recovery liquid to digest matrigel, centrifugally separating the organoids, fixing the organoids with 4% paraformaldehyde, dehydrating the organoids in a 30% sucrose solution for 24 hours, embedding the organoids in 0.C.T ice gel for slicing, treating the slices with dimethylbenzene and gradient ethanol, repairing antigens with a citric acid buffer solution, permeabilizing and blocking the organoids, adding a corresponding primary antibody and a fluorescent-labeled secondary antibody, staining cell nuclei, fixing the glass slide with neutral resin, and photographing and observing under a fluorescent microscope;
s4, after culturing for 48 hours in a 48-well plate, adding 1-50 mu M of Bractoppin into the organoid for two days, adding 20ul of XTT/PMS into the organoid culture medium for further culturing for 2 hours, calculating semi-inhibition concentration values of several organoids after evaluating the vitality of the organoid by using an enzyme-labeling instrument, staining the organoid by using a cell 3D live-dead disease detection kit, marking the live cell and the dead cell as green and red respectively, and photographing by using a fluorescence microscope;
s5, dissolving the Bractoppin in sterile water to obtain 10mM stock solution, subpackaging, storing the solution at-80 degrees, and diluting to corresponding working concentration when in use;
s6, completely lysing protein extraction and western blot cells in RIPA protein lysis buffer, centrifuging to obtain protein solution, measuring protein concentration, adding LDS buffer, heating for denaturation, separating protein by using SDS-PAGE gel, transferring to a membrane, sealing, incubating with primary antibody and secondary antibody, developing with ECL luminescent solution, and photographing;
s7, culturing cells and analyzing the cells of H8910 and SKOV3 in 10% fetal bovine serum, changing a culture medium every 3 days, routinely checking mycoplasma pollution, determining sensitivity of the cells to medicines by using a CCK8 kit, adding 10ml of a CCK8 reagent after the cells grow to 60% in a 96-well plate, and detecting proliferation activity of the cells by using an enzyme-labeled instrument after culturing for 2 hours;
s8, analyzing the influence of the brachypin on cells by using a cell cycle and apoptosis detection kit (C1052, beyotime), collecting the cells by using a scraper, flushing the cells by using PBS after 24 hours of drug treatment, adding the cells into 75% ethanol overnight when carrying out cell cycle analysis, re-suspending the cells as a single cell solution, marking the single cells by using PI, then carrying out cell cycle research by using flow cytometry, staining and detecting the apoptosis by using 5 mu l of annexin V-FITC and 10 mu lPI staining solution, and carrying out flow cytometry after incubating in darkness for 20 minutes;
s9, performing experiments by using a comet detection kit according to the specification, collecting cells by using a rubber scraper after the treatment of the brachypin for 24 hours, cleaning the cells by using PBS, then re-suspending the cells in a soft agarose package and loading the cells on a glass slide coated with agarose, adding alkaline lysate into the gel to dissolve the cells after gel solidification, and performing electrophoresis for 30 minutes in an alkaline environment, and for evaluating the DNA damage level, staining the DNA by using a nucleic acid dye, photographing by using a fluorescence microscope, and determining the distance between the comet tail and the center of a cell nucleus;
s10, analyzing and detecting the functions of DNA non-homologous end connection and homologous recombination, transfecting plasmids into cells by using liposome 3000, inoculating the plasmids into a 24-pore plate, replacing a culture medium after 6 hours, adding 5mM brachopypin, discarding the culture medium, adding a fluorescein substrate, and detecting the activity of the cells by using an enzyme-labeled instrument;
s11, extracting total RNA from organoids by using an RNA extraction kit, generating a sequencing library by using an RNA library building kit, sequencing on a PE150 platform, downloading and cleaning the original data, and comparing the sequences by using HISAT2 software package and Deseq2 software.
2. The method for constructing an ovarian boundary tumor organoid model according to claim 1, wherein: the number of the models is multiple, and the multiple models are all cultured in an in vitro consistent environment.
3. The method for constructing an ovarian boundary tumor organoid model according to claim 1, wherein: the organoid model requires more than 3 months of average in vitro culture time, and the organoid model has a maximum in vitro amplification validation time of more than 8 months.
4. The method for constructing an ovarian boundary tumor organoid model according to claim 1, wherein: in S1, the detergent composition includes: 1X 1640 cell culture solution, Y-27632 (10. Mu.M).
5. The method for constructing an ovarian boundary tumor organoid model according to claim 1, wherein: in the S1, organoid culture fluid composition: 1 Xadvanced DMEM/F12 medium, A8301 (0.5. Mu.M), N-acetyl-L-cysteine (1.25 mM), nicotinamide (5 mM), estradiol (2 Mm), B27 (1X), N2 (1X), ITS (1X), glutamine (1X), HEPES (1X), EGF (50 ng/ml), FGF10 (50 ng/ml), R-spindin (200 ng/ml), noggin (100 ng/ml).
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| CN117625541B (en) * | 2024-01-26 | 2024-04-02 | 零壹人工智能科技研究院(南京)有限公司 | Brain glioma organoid construction method and drug sensitivity detection method |
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