CN115232792B - Culture medium and culture method for pleural fluid source organoid - Google Patents
Culture medium and culture method for pleural fluid source organoid Download PDFInfo
- Publication number
- CN115232792B CN115232792B CN202211136657.1A CN202211136657A CN115232792B CN 115232792 B CN115232792 B CN 115232792B CN 202211136657 A CN202211136657 A CN 202211136657A CN 115232792 B CN115232792 B CN 115232792B
- Authority
- CN
- China
- Prior art keywords
- culture
- pleural effusion
- supernatant
- freeze
- dried powder
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0081—Purging biological preparations of unwanted cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a culture medium and a culture method for pleural effusion source organoids, wherein the culture method comprises the following steps: preparing the breast water supernatant freeze-dried powder, preparing the breast water supernatant freeze-dried powder into a breast water supernatant freeze-dried powder solution, coating the solution in a culture vessel, adding the sediment after the breast water centrifugation into the coated culture vessel for culture after the sediment is resuspended, centrifuging the sediment after the culture is finished to obtain the sediment, mixing the sediment after the sediment is resuspended with a matrix gel, solidifying the sediment, and finally adding a culture medium containing the breast water supernatant freeze-dried powder for culture. According to the invention, the pleural effusion clear freeze-dried powder is added in the culture process, so that the growth state of the organoid is improved, the success rate of the pleural effusion source organoid is improved, and the living environment in the organoid is better simulated.
Description
Technical Field
The invention relates to the technical field of organoid culture, in particular to a culture medium and a culture method for pleural effusion source organoids.
Background
In recent years, with the development of stem cell technology, hans Clever et al established a method for obtaining tissue organoids by 3D culturing stem cells in vitro. Organoids refer to the ability of cells with stem cell potential to be cultured in 3D to form similar tissues of the corresponding organ, and to self-renew and self-organize, maintaining part of the physiological structure and function of the tissue from which they originate. In the past decades, the main model of tumor research is still 2D tumor cell line, but the tumor cell line does not have heterogeneity of tumor cells and characteristics of tumor cells in vivo, and under long-term passage in a laboratory, genomics variation occurs, and cannot reflect the true condition of corresponding tumor; another important model is the human tumor tissue mouse transplantation model (PDX), which can largely maintain the heterogeneity of tumor and is currently used in drug testing and screening of tumor, but still has many problems including low success rate of transplantation, large sample size of tumor and long experimental period. Compared with the prior art, the tumor organoid model can be proliferated indefinitely under in-vitro culture conditions, well maintains tumor heterogeneity, and is suitable for large-scale drug screening.
At present, organoid culture is mainly focused on tumor surgery/puncture samples, organoids with pleural effusion sources are reported less, and pleural effusion samples are easier to obtain clinically than surgery/puncture samples, so that the organoids can be more easily obtained and constructed. There is a report in the literature on a method for removing fibroblasts in a pleural effusion specimen by using a microporous chip and adding pleural effusion supernatant to a culture medium to amplify pleural effusion source organoids, which can improve the success rate of pleural effusion source organoids, but the method requires the pore size of the microporous chip to be 7-20 μm, has high technical requirements and high cost, is not suitable for large-scale application, and the pore plate can only retain and enrich large-size tumor cells or cell clusters, so that the organoids are more lost. The preservation time of the pleural effusion supernatant is short, the pleural effusion supernatant is not easy to preserve, and the pleural effusion supernatant can only be used for culturing specimen organoids from the pleural effusion, and has no universality. In addition, partial cell factors are added into a common in-vitro culture medium, but the in-vivo environment cannot be well reflected, the accuracy and the authenticity of detection data are influenced, the success rate of in-vitro culture of the common culture medium is low, the culture period is relatively long, the culture cost is increased, and the subsequent drug sensitivity experiment process is delayed. In a word, the cost is high, the culture success rate is low, the time consumption is long, and the method is still an important reason that the clinical practicability of the pleural fluid source organoids for in vitro antitumor drug evaluation is low at present.
Disclosure of Invention
In order to solve the above-mentioned deficiencies of the prior art, the present invention aims to provide a culture medium and a culture method for pleural fluid source organoids, so as to solve the problems of high cost, low culture success rate and long time consumption in the prior art.
The technical scheme for solving the technical problems is as follows: there is provided a culture medium for pleural fluid source organoids, comprising: clear freeze-dried powder of pleural effusion, basal medium and additive; the additive comprises the following components in final concentration: human recombinant protein R-Spondin 1, 20-40ng/mL; noggin, 80-120ng/mL; human recombinant protein rhEGF,6-10ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid, 0.8-1.2mM; L-alanyl-L-glutamine, 1 ×; double resistance to cyan chain, 1 ×; n2 additive, 1 ×; b27 additive, 1 ×; N-acetyl-L-cysteine, 0.08-0.1mM; alk inhibitor A83-01,0.3-0.6 μ M;4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, 0.2-0.4 μ M; nicotinamide, 3-6mM.
The beneficial effects of the invention are as follows: the pleural fluid contains a large amount of fibroblasts and tumor cells, the fibroblasts and the tumor cells grow together in a mixed manner in the process of constructing the tumor cell organoids, the fibroblasts usually grow faster than the tumor cells and can inhibit the growth of the tumor cells, and the cultured tumor cell organoids have slower growth speed and lower yield.
The pleural effusion supernatant is freeze-dried to prepare freeze-dried powder, firstly, the prepared pleural effusion supernatant freeze-dried powder can be taken at any time, has long preservation time, stable batch and quantitative property, can be used for culturing different sample organs, does not need to use extra consumables, and greatly saves the cost; secondly, the pleural fluid contains substances for promoting cell attachment such as laminin, fibronectin, collagen type III, serum expansion factor and the like, and after the pleural fluid supernatant freeze-dried powder is added into the culture medium, the adherence of fibroblasts can be promoted, the separation of the fibroblasts and organoids is facilitated, and the purpose of removing the fibroblasts is achieved; finally, the pleural effusion supernatant freeze-dried powder, the basic culture medium and the additives in the culture medium can jointly promote the growth of the organoids, remove impurities, improve the proportion of tumor cells, and further effectively improve the construction efficiency and success rate of the pleural effusion source tumor organoids.
The basic medium is a conventional basic medium, such as Advanced DMEM/F-12 medium.
Further, the concentration of the pleural effusion supernatant freeze-dried powder in the culture medium is 100-200 mug/mL.
Further, the concentration of the pleural effusion supernatant freeze-dried powder in the culture medium is 200 mug/mL.
Further, the additives included the following components in final concentrations: human recombinant protein R-Spondin 1, 30ng/mL; noggin, 100ng/mL; human recombinant protein rhEGF,8ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid, 1mM; L-alanyl-L-glutamine, 1 ×; double resistance to cyan chain, 1 ×; n2 additive, 1 ×; b27 additive, 1 ×; N-acetyl-L-cysteine, 0.09mM; alk inhibitor A83-01, 0.5. Mu.M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, 0.3 μ M; nicotinamide, 5mM.
The method for culturing the pleural effusion source organoids by adopting the culture medium comprises the following steps:
(1) Collecting cancerous pleural effusion, centrifuging, making pleural effusion supernatant into lyophilized powder, and cleaning precipitate;
(2) Dissolving the supernatant freeze-dried powder of the pleural effusion to prepare a supernatant freeze-dried powder solution of the pleural effusion, then adding the solution into a culture vessel for coating, and sucking out the supernatant freeze-dried powder solution of the pleural effusion after coating;
(3) Adding the sediment in the step (1) into a coated culture vessel for culture after the sediment is resuspended, and centrifuging the non-adherent solution after the culture is finished to obtain the sediment;
(4) And (4) resuspending the precipitate in the step (3), mixing with matrigel, solidifying, and finally adding the culture medium for culturing.
The beneficial effects of the invention are as follows: centrifuging cancerous pleural effusion, preparing supernatant into pleural effusion supernatant lyophilized powder, and precipitating to obtain cell precipitate. The chest water supernatant freeze-dried powder has long preservation time, when in use, the chest water supernatant freeze-dried powder is required to be dissolved to form the chest water supernatant freeze-dried powder solution, the chest water supernatant freeze-dried powder solution is used for coating a culture vessel, the adherence of fibroblasts can be promoted more quickly and better, the adherence is slow because of the large organoid volume, the operation is carried out like this, the fibroblasts can adhere to the wall first, and the organoid does not adhere to the wall yet at the moment, the solution without adherence is sucked like this, the centrifugation can separate the fibroblasts from the organoid, and the purpose of removing the fibroblasts is achieved. The method can effectively solve the problems of slow adherence of the fibroblasts, few adherent cells and unclean removal in the method for removing the fibroblasts by a direct sedimentation method.
When the non-adherent solution is centrifuged, the obtained precipitate is mixed with matrigel after being resuspended, and the culture medium containing the pleural effusion clear freeze-dried powder is added for culture after solidification, so as to prepare the pleural effusion source tumor organoid.
In the culture process, trypsin and collagenase are not required to be added to remove the hybrid cells, so that the growth of organoids is not interfered; the method does not need to adopt a microporous chip in the culture process, has low cost and simple operation, can effectively remove fibroblasts, and also solves the problem of much organoid loss in the existing preparation method.
In a word, the pleural effusion supernatant freeze-dried powder is added in the culture process, so that the growth state of the organoid is improved, the problem of slow growth of the pleural effusion source organoid is well solved, the success rate of pleural effusion source organoid culture is greatly improved, the living environment in the organoid is well simulated based on the matching of all components in the culture medium, the culture cost is reduced, and the problems of unstable batch, short storage time and no universality in the prior art in which the pleural effusion supernatant is directly used are solved.
On the basis of the technical scheme, the invention can be further improved as follows:
further, the pleural effusion in the step (1) is derived from the following patients or animals: breast cancer, lung cancer, stomach cancer, esophageal cancer, pancreatic cancer, or peritoneal cancer.
The beneficial effect of adopting the further technical scheme is as follows: the invention has wide application range and can be suitable for culturing the water-in-the-chest organoids of various cancer species.
Further, the cancerous pleural effusion collected in the step (1) is centrifuged, and after the filtration and impurity removal through a filter membrane, the cancerous pleural effusion is inactivated and finally freeze-dried to prepare the pleural effusion clear freeze-dried powder, and the specific process is as follows: centrifuging the collected cancerous pleural effusion at 4 deg.C and 1300rpm, collecting pleural effusion supernatant, filtering pleural effusion supernatant with 0.22 μm filter membrane, removing impurities, inactivating in 56 deg.C water bath for 30min, freezing at-80 deg.C for solidification, vacuumizing, sublimation drying, and removing ice crystal to obtain lyophilized powder.
Further, the method also comprises the following steps: after the precipitate in the step (1) is resuspended, adding erythrocyte lysate to remove erythrocytes, and the specific process comprises the following steps: and (2) after the sediment in the step (1) is resuspended, adding erythrocyte lysate with the volume 3 times that of the cell suspension, uniformly mixing, incubating for 15min on ice, then centrifuging for 10min at the temperature of 4 ℃ at 450 Xg, collecting cells, sucking and removing supernatant, carrying out the next operation after the sediment is resuspended, and if more erythrocytes exist in the obtained cell sediment, continuing to repeat the steps.
Further, dissolving the clear chest water freeze-dried powder in the step (2) to prepare 0.8-2mg/mL of the clear chest water freeze-dried powder solution, and then adding the solution into a culture vessel to coat for 15-40min at 35-40 ℃. Preferably, the concentration of the clear freeze-dried powder solution of the breast water is 1mg/mL, the coating temperature is 37 ℃, and the coating time is 30min.
The beneficial effects of adopting the further technical scheme are as follows: the culture vessel is coated with high-concentration pleural effusion supernatant lyophilized powder solution in advance, so that the adhesion of fibroblasts can be promoted more quickly and better, and the removal of the fibroblasts is facilitated.
Further, the culture conditions in the step (3) are as follows: culturing at 37 deg.C for 15-30min, preferably 20min.
The beneficial effects of adopting the further technical scheme are as follows: and (3) resuspending the precipitate to obtain a cell suspension, adding the cell suspension into a coated culture vessel, and culturing at 37 ℃ for 15-30min to make the fibroblasts adhere to the wall, wherein the culturing time is especially 20min, and the cell suspension has a good adherence effect and low cost.
Drawings
FIG. 1 is a morphological diagram of the organoid prepared in example 1.
FIG. 2 is a morphological diagram of organoids prepared in comparative example 1.
FIG. 3 is a morphological diagram of the organoid prepared in comparative example 2.
FIG. 4 is a morphological diagram of the organoid prepared in comparative example 3.
FIG. 5 shows the success rate of culturing pleural effusion organoids of different cancer species in comparative example 5.
Detailed Description
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1:
a culture medium for pleural effusion source organoid comprises pleural effusion clear lyophilized powder, basal culture medium and additive;
the additive comprises the following components in final concentration: human recombinant protein R-Spondin 1, 30ng/mL; noggin, 100ng/mL; human recombinant protein rhEGF,8ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid, 1mM; L-alanyl-L-glutamine, 1 ×; double resistance to cyan chain, 1 ×; n2 additive, 1 ×; b27 additive, 1 ×; N-acetyl-L-cysteine, 0.09mM; alk inhibitor A83-01, 0.5. Mu.M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, 0.3 μ M; nicotinamide, 5mM;
the basic culture medium is Advanced DMEM/F-12 culture medium;
the concentration of the pleural effusion supernatant freeze-dried powder in the culture medium is 200 mug/mL.
The method for culturing the pleural effusion source organoids by adopting the culture medium comprises the following steps:
(1) Preparing clear freeze-dried powder of breast water: collecting breast cancer pleural effusion sample of a patient in a sterile environment, centrifuging at 1300rpm, collecting pleural effusion supernatant, filtering the pleural effusion supernatant with 0.22 μm filter membrane to remove impurities, inactivating in 56 deg.C water bath for 30min, freezing at-80 deg.C for solidification, vacuumizing, sublimation drying, and removing ice crystal to obtain pleural effusion supernatant lyophilized powder;
the collected sediment after the pleural effusion centrifugation is washed for 3 times by PBS;
(2) Removing red blood cells: the cell sediment obtained in the step (1) is red, which indicates that more red blood cells are needed to be removed, and the specific process is as follows: resuspending the cell precipitate to form a cell suspension, adding erythrocyte lysate with the volume 3 times that of the cell suspension into the cell suspension, gently swirling or turning over and mixing uniformly, then incubating on ice for 15min, gently swirling and mixing uniformly twice during the period, then centrifuging at 4 ℃ and 450 Xg for 10min to collect cells, carefully sucking and removing supernatant, and repeating the operation until the cell precipitate does not show red;
(3) Removing fibroblasts: adding 1mg of breast water supernatant freeze-dried powder into 1mL of sterile PBS to prepare a high-concentration (1 mg/mL) breast water supernatant freeze-dried powder solution, then adding the solution into a culture dish to be coated, standing at 37 ℃ for 30min, sucking out the breast water supernatant freeze-dried powder solution, adding the cells collected in the step (2) into the coated culture dish after being resuspended, placing the culture dish in a 37 ℃ incubator to be cultured for 20min to allow fibroblasts to adhere to the wall, carefully sucking the non-adherent cells on the upper layer and centrifuging to obtain the organoid without the fibroblasts;
(4) Counting the cell pellet of step (3) by a cell counting plate, resuspending the cell pellet in Matrigel containing 50% of Matrigel according to the counting result, dropping the cell pellet containing 5000 cells per 50. Mu.L into a well of a 24-well plate, and then, 5% CO at 37 ℃% 2 Standing for 5min under the condition, coagulating the gel solution, adding 1mL of the above culture medium containing the supernatant lyophilized powder of pleural effusion into each well, and 5% of CO at 37 deg.C 2 Culturing under the condition, periodically photographing and counting the number and the surface area of organoids.
Example 2:
example 2 differs from example 1 in that the concentration of the pleural effusion supernatant lyophilized powder in the culture medium is 100. Mu.g/mL.
Example 3:
the difference between the embodiment 3 and the embodiment 1 is that the concentration of the clear pleural effusion lyophilized powder solution in the step (3) is 0.8mg/mL, and the coating time is 40min; the culture time is 30min.
Example 4:
example 4 is different from example 1 in that, in step (3), the concentration of the clear and lyophilized powder solution of the breast water is 2mg/mL, and the coating time is 15min; the culture time is 15min.
Example 5:
example 5 differs from example 1 in the content of additives, in particular: the additive comprises the following components in final concentration: human recombinant protein R-Spondin 1, 20ng/mL; noggin, 80ng/mL; human recombinant protein rhEGF,6ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid, 0.8mM; L-alanyl-L-glutamine, 1 ×; double resistance of the green chain, 1 ×; n2 additive, 1 ×; b27 additive, 1 ×; N-acetyl-L-cysteine, 0.08mM; alk inhibitor A83-01, 0.3. Mu.M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, 0.2 μ M; nicotinamide, 3mM.
Example 6:
example 6 differs from example 1 in the content of additives, specifically: the additive comprises the following components in final concentration: human recombinant protein R-Spondin 1, 40ng/mL; noggin, 120ng/mL; human recombinant protein rhEGF,10ng/mL; 4-hydroxyethyl piperazine ethanesulfonic acid, 1.2mM; L-alanyl-L-glutamine, 1 ×; double resistance to cyan chain, 1 ×; n2 additive, 1 ×; b27 additive, 1 ×; N-acetyl-L-cysteine, 0.1mM; alk inhibitor A83-01, 0.6. Mu.M; 4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, 0.4 μ M; nicotinamide, 6mM.
Example 7:
the breast cancer supernatant freeze-dried powder in the example 7 is also freeze-dried powder prepared from breast cancer supernatant of a patient, but the difference from the breast cancer supernatant freeze-dried powder in the example 1 is as follows: the cultured organoid is the organoid from the pleural effusion of lung cancer of a patient, namely the pleural effusion of lung cancer of the patient is centrifuged, the obtained precipitate is washed 3 times by PBS and is used in the steps (2) to (4), the processes from the step (2) to the step (4) are the same as in the example 1, and the culture medium is also the same.
Example 8:
in example 8, the breast cancer supernatant lyophilized powder is also a lyophilized powder prepared from breast cancer supernatant of a patient, but the difference from example 1 is as follows: the cultured organoids were those derived from gastric cancer pleural effusion of patients, i.e., the gastric cancer pleural effusion of patients was centrifuged, and the obtained precipitates were washed 3 times with PBS and then used in the steps (2) to (4), and the procedures of the steps (2) to (4) were the same as those of example 1.
Example 9:
the breast cancer supernatant freeze-dried powder in the example 9 is also freeze-dried powder prepared from breast cancer supernatant of a patient, but the difference from the breast cancer supernatant freeze-dried powder in the example 1 is as follows: the cultured organoids are those derived from the pleural fluid of esophageal cancer of a patient, i.e., the pleural fluid of esophageal cancer of a patient is centrifuged, and the obtained precipitate is washed 3 times with PBS and then used in the steps (2) to (4), and the procedures of the steps (2) to (4) are the same as those in example 1.
Example 10:
in example 10, the breast cancer supernatant lyophilized powder is also a lyophilized powder prepared from breast cancer supernatant of a patient, but the difference from example 1 is as follows: the cultured organoids were those derived from the pleural fluid of pancreatic cancer in a patient, i.e., the pleural fluid of pancreatic cancer in a patient was centrifuged, and the resulting pellet was washed 3 times with PBS and then used in the steps (2) to (4), and the procedures of the steps (2) to (4) were the same as in example 1.
Example 11:
the breast cancer supernatant freeze-dried powder in the example 11 is also freeze-dried powder prepared from breast cancer supernatant of a patient, but the difference from the breast cancer supernatant freeze-dried powder in the example 1 is as follows: the cultured organoids were those derived from the pleural effusion of the peritoneal cancer of the patient, i.e., the peritoneal cancer pleural effusion of the patient was centrifuged, and the obtained pellet was washed 3 times with PBS and then used in the steps (2) to (4), and the procedures of the steps (2) to (4) were the same as in example 1.
Comparative example 1:
comparative example 1 differs from example 1 in that the removal of fibroblasts was not performed.
The organoids prepared as described above were examined as follows, and using example 1 as an example, the organoid morphology map prepared in example 1 is shown in fig. 1, and the organoid morphology map prepared in comparative example 1 is shown in fig. 2.
As can be seen from fig. 1 and 2, the number of organoids cultured for 10 days after the fibroblast removal process was higher than that of the organoids without removing fibroblasts, the number of adherently differentiated cells in fig. 1 was also less than that in fig. 2, and the organoid size was also larger than that in comparative example 1 after removing fibroblasts.
The total organoid number and average surface area results after 10 days in culture are shown in table 1:
TABLE 1 organoid numbers and average surface areas obtained by culturing according to example 1 and comparative example 1
As can be seen from Table 1, the fibroblast removal process was performed in example 1, and the number of organoids cultured for 10 days was significantly higher than that of non-removed fibroblasts, and the average surface area of organoids in example 1 was also significantly higher than that in comparative example 1.
Comparative example 2:
comparative example 2 differs from example 1 in that, when fibroblasts were removed, the culture dish was directly resuspended and then added to the culture dish without coating with clear freeze-dried powder on breast water, the culture dish was placed in an incubator at 37 ℃ for 20min, and the upper non-adherent cells were carefully aspirated and centrifuged.
The organoid morphology map prepared in comparative example 2 is shown in FIG. 3; the total organoid number and average surface area results after 10 days in culture are shown in table 2.
As can be seen from fig. 1 and 3, when the culture dish is coated with the pleural effusion supernatant lyophilized powder solution to remove fibroblasts, the number of organoids after 10 days of culture is higher than that of organoids without the pleural effusion supernatant lyophilized powder solution, the number of adherently differentiated cells in fig. 1 is less than that in fig. 3, and after the fibroblasts are removed, the size of organoids is larger than that in comparative example 2.
TABLE 2 organoids number and average surface area obtained by culturing according to example 1 and comparative example 2
As shown in Table 2, in example 1, the number of organoids in the culture dish coated with the pleural effusion supernatant lyophilized powder solution is higher than that in the culture dish not coated with the pleural effusion supernatant lyophilized powder solution after 10 days of culture, and the average surface area of organoids in example 1 is also obviously higher than that in comparative example 2.
Comparative example 3:
comparative example 3 is different from example 1 in that, in the removal of fibroblasts, fibroblasts having a small size were removed by filtration using a microporous chip having a pore size of 20 μm, and the tumor organoids which were not filtered were collected and cultured.
The organoid morphology map prepared in comparative example 3 is shown in FIG. 4; the total organoid number and average surface area results after 10 days in culture are shown in Table 3.
As can be seen from fig. 1 and 4, compared to comparative example 3 in which fibroblasts were removed by the microporous chip, in example 1, fibroblasts were removed by inclusion in a culture dish using the freeze-dried powder of the supernatant of the pleural effusion, and after 10 days of culture, the cells differentiated by adherence and organoids were close in size, but the organoids number was much higher than in comparative example 3 in which fibroblasts were removed by the microporous chip.
TABLE 3 organoids number and average surface area obtained by culturing according to example 1 and comparative example 3
As shown in Table 3, in example 1, the culture dish is coated with the pleural effusion supernatant lyophilized powder solution to remove fibroblasts, and after 10 days of culture, the number of organoids is obviously higher than that of comparative example 3 in which the microcellular chip is used to remove fibroblasts.
Comparative example 4:
the difference between the comparative example 4 and the example 1 is that the adding proportion of the pleural effusion supernatant freeze-dried powder in the culture medium is different, and the influence results of adding the pleural effusion supernatant freeze-dried powder with different concentrations on the total amount and the average surface area of the obtained organoids are shown in a table 4.
TABLE 4 Effect of different concentrations of pleural effusion lyophilized powder on total number and average surface area of organoids
Comparative example 5:
comparative example 5 is different from example 1 and examples 7-11 in that the breast cancer pleural effusion clear lyophilized powder of a patient is not added in the culture medium, and the culture success rate of different cancer species pleural effusion organoids is shown in figure 5.
As can be seen from FIG. 5, after 10 days of culture, the breast cancer breast water supernatant lyophilized powder group is added into the culture medium, and the success rate of breast cancer, lung cancer, gastric cancer, esophageal cancer, pancreatic cancer and peritoneal cancer organ culture is obviously superior to that of breast cancer breast water supernatant lyophilized powder group which is not added, which shows that the breast water supernatant lyophilized powder can be used for culturing different organs and has universality.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A culture method of pleural fluid source organoids is characterized by comprising the following steps:
(1) Collecting breast cancer hydrothorax, centrifuging, making hydrothorax supernatant into lyophilized powder, and cleaning precipitate;
(2) Dissolving the supernatant freeze-dried powder of the pleural effusion to prepare a supernatant freeze-dried powder solution of the pleural effusion, then adding the solution into a culture vessel for coating, and sucking out the supernatant freeze-dried powder solution of the pleural effusion after coating;
(3) Adding the sediment in the step (1) into a coated culture vessel for culture after the sediment is resuspended, and centrifuging the non-adherent solution after the culture is finished to obtain the sediment;
(4) Resuspending the precipitate in the step (3), then mixing with matrigel, solidifying, and finally adding a culture medium for the pleural fluid source organoids for culture;
the culture medium for pleural fluid-derived organoids, comprising: the pleural effusion supernatant freeze-dried powder, a basic culture medium and an additive; the additive comprises the following components in final concentration: human recombinant protein R-Spondin 1, 20-40ng/mL; noggin, 80-120ng/mL; human recombinant protein rhEGF,6-10ng/mL; 4-hydroxyethylpiperazine ethanesulfonic acid, 0.8-1.2mM; L-alanyl-L-glutamine, 1 ×; double resistance to cyan chain, 1 ×; n2 additive, 1 ×; b27 additive, 1 ×; N-acetyl-L-cysteine, 0.08-0.1mM; alk inhibitor A83-01,0.3-0.6 μ M;4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, 0.2-0.4 μ M; nicotinamide, 3-6mM.
2. The culture method according to claim 1, wherein the concentration of the supernatant lyophilized powder of pleural effusion in the culture medium is 100-200 μ g/mL.
3. The culture method according to claim 2, wherein the concentration of the supernatant lyophilized powder of pleural effusion in the culture medium is 200 μ g/mL.
4. The cultivation process according to any one of claims 1 to 3, wherein the additives comprise the following components in final concentrations: human recombinant protein R-Spondin 1, 30ng/mL; noggin, 100ng/mL; human recombinant protein rhEGF,8ng/mL; 4-hydroxyethyl piperazine ethanesulfonic acid, 1mM; L-alanyl-L-glutamine, 1 ×; double resistance to cyan chain, 1 ×; n2 additive, 1 ×; b27 additive, 1 ×; N-acetyl-L-cysteine, 0.09mM; alk inhibitor A83-01,0.5 μ M;4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole, 0.3 μ M; nicotinamide, 5mM.
5. The culture method according to claim 1, wherein the breast cancer pleural effusion collected in the step (1) is centrifuged, and after the filtration and impurity removal through a filter membrane, the breast cancer pleural effusion is inactivated and finally freeze-dried to prepare the breast cancer pleural effusion clear freeze-dried powder.
6. The culture method according to claim 1, further comprising: and (2) after the precipitate in the step (1) is resuspended, adding erythrocyte lysate and removing erythrocytes.
7. The culture method according to claim 1, wherein the pleural effusion supernatant lyophilized powder is dissolved in step (2) to obtain a pleural effusion supernatant lyophilized powder solution of 0.8-2mg/mL, and then the solution is added into a culture vessel to be coated for 15-40min at 35-40 ℃.
8. The culture method according to claim 1, wherein the culture conditions in step (3) are: culturing at 37 deg.C for 15-30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211136657.1A CN115232792B (en) | 2022-09-19 | 2022-09-19 | Culture medium and culture method for pleural fluid source organoid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211136657.1A CN115232792B (en) | 2022-09-19 | 2022-09-19 | Culture medium and culture method for pleural fluid source organoid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115232792A CN115232792A (en) | 2022-10-25 |
| CN115232792B true CN115232792B (en) | 2023-01-03 |
Family
ID=83681350
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211136657.1A Active CN115232792B (en) | 2022-09-19 | 2022-09-19 | Culture medium and culture method for pleural fluid source organoid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115232792B (en) |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108707582A (en) * | 2018-05-24 | 2018-10-26 | 上海赫佰生物医药科技有限公司 | A kind of high-efficient culture method and its application of the tumour cell in body fluid source |
| CN110373388A (en) * | 2019-04-24 | 2019-10-25 | 博奥生物集团有限公司 | A kind of culture medium and organoid cultural method for organoid culture |
| CN110129270B (en) * | 2019-05-27 | 2021-02-09 | 创芯国际生物科技(广州)有限公司 | Culture medium, culture method and drug sensitivity test method for pleural and peritoneal fluid organs |
| WO2021021503A1 (en) * | 2019-07-26 | 2021-02-04 | The Children's Medical Center Corporation | Use of alveolar or airway organoids for the treatment of lung diseases and disorders |
| EP4073232A4 (en) * | 2019-12-12 | 2024-01-24 | The Walter and Eliza Hall Institute of Medical Research | Organoid cultures |
| CN113881635A (en) * | 2021-11-11 | 2022-01-04 | 浙江省肿瘤医院 | Culture medium and culture method for pleural mesothelioma organoid |
| CN114231491B (en) * | 2021-12-23 | 2024-06-04 | 北京基石生命科技有限公司 | Method for preparing hydrogel personalized culture tumor organoids by using autologous chest and ascites of tumor patient |
-
2022
- 2022-09-19 CN CN202211136657.1A patent/CN115232792B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN115232792A (en) | 2022-10-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2023274338A1 (en) | Culture medium, method, and reagent kit for rapidly culturing tumor organoids | |
| CN114317443B (en) | Breast cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| CN114292816B (en) | Lung cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| CN111411083B (en) | Culture medium and culture method for stomach cancer organoid | |
| CN108103013B (en) | Enzyme digestion method primary culture and identification method for smooth muscle cells of esophageal-gastric junction | |
| CN111197030A (en) | Method for culturing bladder cancer organoid in vitro | |
| CN108504625B (en) | Mouse fibroblast and application thereof | |
| CN112522201A (en) | Culture medium and culture method for bladder cancer organoid | |
| CN106801032A (en) | The construction method of people's amnioic epithelium stem cell bank | |
| CN117004572A (en) | Construction method and application of patient-derived transplanted tumor organoid model PDXO | |
| CN112126618B (en) | Method for obtaining human gallbladder stem cells and long-term in vitro culture | |
| CN111235100B (en) | Culture method of human umbilical cord blood mesenchymal stem cells | |
| CN116836934B (en) | Osteosarcoma organoid culture solution, culture reagent combination and culture method | |
| CN115232792B (en) | Culture medium and culture method for pleural fluid source organoid | |
| CN111321120B (en) | Separation and culture method of circulating tumor cell line of mouse renal clear cell carcinoma and circulating tumor cell of human-derived renal clear cell carcinoma | |
| CN113736737B (en) | Primary glioma-related fibroblast culture method | |
| CN114231491B (en) | Method for preparing hydrogel personalized culture tumor organoids by using autologous chest and ascites of tumor patient | |
| CN111961640A (en) | Construction method and culture system of three-dimensional differentiation model of urinary renal stem cells | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| CN108048390B (en) | Method for preparing vascular endothelial cells and special kit thereof | |
| CN116536265A (en) | Special organoid culture medium for liver cancer, culture method and passage method | |
| CN116286655A (en) | Culture medium suitable for culturing multiple solid tumor organoids and culture method thereof | |
| CN112760289B (en) | Special culture medium for breast cancer organoid and 3D culture method | |
| CN112430568B (en) | Method for feeding epithelial source organoid by umbilical cord mesenchymal stem cells | |
| CN110607279B (en) | 3D culture system of primary tumor cells, and culture method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |