CN115975939A - Culture medium for cervical cancer organoid, and culture method and application thereof - Google Patents
Culture medium for cervical cancer organoid, and culture method and application thereof Download PDFInfo
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Abstract
The invention relates to a culture medium for cervical cancer organoids, comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632, a83-01, epidermal growth factor, fibroblast growth factor 10, keratinocyte growth factor, glutaMAX and nicotinamide. The invention also relates to a culture method of the cervical cancer organoids. By using the cervical cancer organoid culture medium, the cervical cancer organoid can be effectively and rapidly amplified, so that the amplified organoid keeps the pathological characteristics of a patient, the culture success rate and the amplification rate of the cervical cancer organoid are improved, and a research basis can be provided for the personalized treatment of the patient.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium for cervical cancer organoids and a method for culturing the cervical cancer organoids by using the culture medium.
Background
Cervical cancer is a malignant tumor that originates at the cervical region and is the most common malignant tumor of the female reproductive system. Cervical cancer is currently ranked fourth in female malignancies worldwide. The cervical cancer is higher than 30-55 years old, and the incidence rate is only behind breast cancer in China. Due to the popularization of cervical cancer screening and the popularization of HPV vaccines, cervical cancer becomes a preventable disease to a large extent, but the 5-year survival rate of the cervical cancer is only about 60 percent at present. For early-stage non-metastatic cervical cancer, surgery and chemoradiotherapy are the main treatment means, but for metastatic or recurrent cervical cancer, the traditional treatment method does not achieve satisfactory curative effect. With the use of anti-angiogenesis drugs, immune checkpoint inhibitors and other targeted therapies and immunotherapeutic drugs, the survival time of this part of patients is significantly prolonged, but the final curative effect is not yet achieved, and it is urgently needed to explore new drug targets and guide personalized therapies by means of tumor primary cells as research models.
Two-dimensional cell culture is mostly adopted in the traditional clinical drug sensitivity detection. However, the two-dimensional cultured cells only simulate the tissue physiological conditions to a limited extent, lack the real tissue structure in vivo, easily cause low differentiation level and loss of cell physiological functions, and further cause the obtained experimental results to be difficult to predict the clinical practical results. Organoids, belonging to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation capacity. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional forms of the organs. Under certain induction conditions in vitro, the stem cells can self-organize to form a mini-structure with a diameter of only a few millimeters. Tumor organoids are miniature 3D tumor cell models grown in the laboratory from primary tumors taken from patients. The tumor organoids highly mimic the characteristics of the source tumor tissue, retain inter-individual tumor heterogeneity, and can be used for functional testing, such as drug screening and individualized precision therapy.
Currently, the cervical cancer organoid culture method mostly adopts expensive protein factors such as R-spondin-1, WNT3A and Noggin and the like, so that the organoid culture cost is higher; and the technical operation is complex and the technical difficulty is high, so that the large-scale commercial application of the technology is limited. Therefore, there is a need to develop a low-cost, simple and high-success organoid culture method and medium.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture medium and a culture method for rapidly amplifying cervical cancer organoids in vitro.
One aspect of the present invention provides a culture medium for a cervical cancer organoid, said culture medium comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from the group consisting of N2 and B27, hepatocyte growth factor, SB202190, Y27632, a83-01, epidermal growth factor, fibroblast growth factor 10, keratinocyte growth factor, glutaMAX and nicotinamide. Wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,
wherein the content of the first and second substances,
R 1 selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R 6 Substituted aryl (e.g., phenyl, naphthyl, and the like), arylC 1-C6 alkyl (e.g., benzyl, and the like), and heteroaryl (e.g., benzyl, and the like)Thienyl, etc.);
R 2 and R 3 Each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocyclylC 1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, and the like);
R 6 selected from the group consisting of halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).
In a preferred embodiment, the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
wherein the content of the first and second substances,
R 1 selected from C1-C6 alkyl, optionally substituted with 1-2 independent R 6 Substituted phenyl, optionally substituted with 1-2 independent R 6 Substituted thienyl, and optionally substituted with 1-2 independent R 6 Substituted benzyl, R 1 More preferably optionally substituted with 1-2 independent R 6 Substituted phenyl;
R 5 selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 More preferably hydrogen;
R 6 each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, R 6 More preferably fluorine, methyl or trifluoromethyl.
Preferably, the MST1/2 inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt, or solvate thereof.
Most preferably, the MST1/2 kinase inhibitor of the invention is compound 1.
In an embodiment of the invention, the content of each component in the medium of the invention satisfies any one or more or all of the following:
(1) The concentration of the MST1/2 kinase inhibitor is preferably 2.5-10 mu M;
(2) The volume ratio of the B27 or N2 cell culture additive to the culture medium is 1;
(3) The concentration of the hepatocyte growth factor is preferably 5-40 ng/mL;
(4) The concentration of SB202190 is preferably 200-1000 nM;
(5) The concentration of Y27632 is preferably 2.5-10 μ M;
(6) The concentration of A83-01 is preferably 200-1000 nM;
(7) The concentration of the epidermal growth factor is preferably 1-40 ng/mL;
(8) The concentration of the fibroblast growth factor 10 is preferably 10-100 ng/mL;
(9) The concentration of the keratinocyte growth factor is preferably 2-40 ng/mL;
(10) The volume ratio of GlutaMAX to the culture medium is preferably 1;
(11) The concentration of nicotinamide is preferably 1 to 10mM.
In an embodiment of the invention, said medium further comprises a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin.
In a preferred embodiment, the streptomycin concentration ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from streptomycin/penicillin, the penicillin concentration ranges from 25 to 400U/mL, the concentration ranges from 0.25 to 4. Mu.g/mL when the antibiotic is selected from amphotericin B, and the concentration ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from Primocin.
The invention also provides a culture method of the cervical cancer organoid. In the method for culturing a cervical cancer organoid of the present invention, the cervical cancer organoid is cultured using the cervical cancer organoid culture medium of the present invention.
The cervical cancer organoid culture method of the present invention comprises the following steps.
1. Separating a sample from a cervical cancer solid tumor tissue to obtain cervical cancer primary cells. The treatment process comprises the following steps:
(1) Separating a cervical cancer tissue sample, adding a basic culture medium and tissue digestive juice (the adding amount of the tissue digestive juice is about 10mL per 1g of tumor tissue) in a proportion of 1:1, placing the mixture in a constant-temperature shaking table for digestion, wherein the digestion temperature is 4-37 ℃, the rotation speed of the shaking table is 200-300 rpm, and the digestion time is 3-6 hours;
(2) After digestion, centrifuging and discarding supernatant, wherein the centrifugation speed is 1200-1600 rpm, and the centrifugation time is 2-6 minutes.
Wherein the basal medium formulation comprises an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin. The formula of the tissue digestive fluid comprises a 1640 culture medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNase (50-100U/mL), hyaluronidase (0.5-1 mg/mL), calcium chloride (1-5 mM) and bovine serum albumin BSA (5-10 mg/mL).
2. Preparing the cervical cancer organoid culture medium, and culturing the cervical cancer primary cells obtained in the step.
Resuspending and counting the cervical cancer primary cells obtained in the step 1 by using the cervical cancer organoid culture medium of the invention, and diluting the cell density to 5-10 × 10 5 And (2) taking out the diluted cell suspension, adding the diluted cell suspension into Matrigel with the same volume, uniformly mixing, then inoculating the mixture into a porous plate, putting the inoculated porous plate into an incubator for 30-60 minutes, and adding a cervical cancer organoid culture medium for amplification culture after the Matrigel is completely solidified.
In other aspects of the present invention, there is also provided a method for evaluating or screening a drug for treating cervical cancer, comprising the steps of:
(1) The cervical cancer organoid is cultured by using the cervical cancer organoid culture method;
(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;
(3) Adding the diluted medicine to the cervical cancer organoids cultured in (1); and
(4) Organoid size or organoid viability detection is performed.
The beneficial effects of the invention include:
(1) The success rate of the cervical cancer tissue source organoid culture is improved and reaches more than 85 percent;
(2) Ensuring that the cervical cancer organs subjected to in vitro primary culture can keep the pathological characteristics of patients;
(3) The amplification efficiency is high, the cervical cancer organoid can be rapidly cultured, and the amplified cervical cancer organoid can be continuously passed;
(4) The culture cost is controllable, and expensive Wnt agonist, R-spondin family protein and Noggin protein do not need to be added into the culture medium;
(5) The cervical cancer organoids obtained by the technology are large in number, and the technology is suitable for screening candidate compounds in high flux and providing high-flux medicine in-vitro sensitivity function tests for patients.
Drawings
FIGS. 1A-1K are graphs showing the effect of different concentrations of factors added to cervical cancer organoid media of the present invention on cervical cancer organoid proliferation.
FIGS. 2A to 2D are photographs for observing, with a microscope, a cervical cancer organoid cultured using the cervical cancer organoid medium of the present invention, wherein FIG. 2A shows a photograph of organoid culture obtained from a specimen OC1 for 5 days; FIG. 2B shows a photograph of organoids obtained from sample OC1 after 14 days of culture; FIGS. 2C and 2D show photographs of different fields of view obtained from sample OC2 after 7 days of organoid culture.
FIG. 3 shows the results of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoids obtained by culturing the sample OC4 with the cervical cancer organoid culture medium of the present invention.
FIGS. 4A and 4B are comparative results of culture of a cervical cancer organoid using the cervical cancer organoid medium of the present invention and a prior art medium, in which FIG. 4A shows photographs after 10 days of culture with the COM medium of the present invention; FIG. 4B shows a photograph after 10 days of culture with the literature medium ROM.
Fig. 5 is a graph showing the results of a drug concentration sensitivity test on cervical cancer organoids cultured using the cervical cancer organoid culture medium of the present invention.
Detailed Description
For a better understanding of the present invention, the present invention is further described below with reference to the following examples and the accompanying drawings. The following examples are intended to illustrate the invention and are not intended to limit it.
[ preparation example of MST1/2 kinase inhibitor ]
In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly down-regulates MST1/2 signaling. In general, MST1/2 kinase inhibitors, for example, bind to and reduce the activity of MST1/2 kinase. Due to the structural similarity of MST1 and MST2, MST1/2 kinase inhibitors may also be compounds that bind to and reduce the activity of MST1 or MST2, for example.
Preparation of MST1/2 kinase inhibitor Compound 1
4- ((7- (2,6-difluorophenyl) -5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl) amino) benzene
Sulfonamide 1
Methyl 2-amino-2- (2,6-difluorophenyl) acetate (A2): after 2-amino-2- (2,6-difluorophenyl) acetic acid (2.0 g) was added to the round bottom flask, methanol (30 ml) was added followed by thionyl chloride (1.2 ml) dropwise under ice-bath. The reaction system was reacted at 85 ℃ overnight. After the reaction was complete, the solvent was evaporated to dryness under reduced pressure to give a white solid which was used directly in the next step.
Methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (A3): to a round bottom flask was added methyl 2-amino-2- (2,6-difluorophenyl) acetate (2 g) followed by acetone (30 ml) and potassium carbonate (2.2 g) then the system was cooled to-10 ℃ with an ice salt bath followed by the slow addition of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressure silica gel column chromatography to obtain Compound A3.LC/MS: m + H359.0.
2-chloro-7- (2,6-difluorophenyl) -7,8-dihydropteridin-6 (5H) -one (A4): to a round bottom flask was added methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (2.5 g) followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction was stirred at 60 ℃ for two hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the obtained product was neutralized to be alkaline with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A4.LC/MS: m + H297.0.
2-chloro-7- (2,6-difluorophenyl) -5,8-dimethyl-7,8-dihydropteridin-6 (5H) -one (A5): 2-chloro-7- (2,6-difluorophenyl) -7,8-dihydropteridin-6 (5H) -one (2 g) and N, N-dimethylacetamide (10 mL) were added to a round bottom flask, cooled to-35 deg.C, iodomethane (0.9 mL) was added followed by sodium hydride (615 mg) and the reaction was stirred for an additional two hours. After the reaction, water was added to quench, ethyl acetate was used for extraction, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A5.LC/MS: m + H325.0.
4- ((7- (2,6-difluorophenyl) -5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl) amino) benzenesulfonamide (1): into a round bottom flask was added 2-chloro-7- (2,6-difluorophenyl) -5,8-dimethyl-7,8-dihydropteridin-6 (5H) -one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 ml). The reaction was stirred at 120 ℃ overnight. After the reaction is finished, filtering, and washing by methanol and ether to obtain the compound 1.LC/MS: m + H461.1.
2. Preparation of other MST1/2 inhibitor compounds of the invention
Other MST1/2 inhibitor compounds of the invention were synthesized in analogy to compound 1 and their structural and mass spectral data are shown in the table below.
Example 1 Effect of factors added to cervical cancer organoid Medium on proliferation of cervical cancer organoids
(1) Preparation of cervical cancer organoid culture medium
First, a basal medium containing an initial medium is prepared. The starting medium may be selected from DMEM/F12, DMEM, F12 or RPMI-1640, commonly used in the art. In this example, the basic medium formulation is: DMEM/F12 medium (from Corning) + 100. Mu.g/mL Primocin (from InvivoGen, 0.2% (v/v), commercial product concentration 50 mg/mL).
Different additives (see table 1) are added into the basal culture medium respectively to prepare the cervical cancer organoid culture medium containing different additive components.
(2) Isolation and processing of cervical cancer primary cells
1 sample selection
Cervical cancer solid tumor tissue samples (intraoperatively) were obtained from patients by medical professionals at specialized medical institutions, who all signed informed consent. Intraoperative sample 5-10mm 3 And the commercial tissue preservation solution (manufacturer: miltenyi Biotec) is adopted for storage and transportation.
2 Material preparation
And (3) sterilizing the surfaces of a 15mL sterile centrifuge tube, a pipette, a 10mL pipette, a sterile gun head and the like, and then placing the sterilized surfaces into an ultra-clean workbench for ultraviolet irradiation for 30 minutes. Basal medium was removed from the 4 ℃ freezer 30 minutes earlier and tissue digest was removed from the-20 ℃ freezer 30 minutes earlier.
The formula of the tissue digestive juice comprises the following components: 1640 Medium (Corning, 10-040-CVR), collagenase II (2 mg/mL), collagenase IV (2 mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3 mM), bovine serum albumin BSA (10 mg/mL).
Collagenase ii, collagenase iv, dnase, hyaluronidase mentioned above were all purchased from Sigma company; calcium chloride and BSA were purchased from Biotechnology engineering (Shanghai) Inc.
3 separation of samples
3.1 taking tissue samples in a Petri dish in a clean bench, removing blood-carrying tissue, rinsing 2 times with a basal medium, transferring the tissue to another Petri dish, mechanically separating with a sterile scalpel, dividing the tissue block into 1 x 2 x 1mm 3 Size;
3.2 sucking the cut intraoperative tissue into a 15mL centrifuge tube, adding 5mL basic culture medium, uniformly mixing, and centrifuging at 1500rpm for 3 minutes;
3.3 abandoning the supernatant, adding the basal medium and the tissue digestive juice in a proportion of 1:1 (note: the adding amount of the tissue digestive juice is 1g of tumor tissue and about 10mL of the tissue digestive juice), marking the name and the number of the sample, sealing the sample by a sealing film, digesting the sample in a shaking table (ZQLY-180N) at 300rpm at 37 ℃, observing whether the digestion is finished every 30 minutes, and judging that no visible particles exist;
3.4 after the digestion is finished, filtering out undigested tissue agglomerates through a 70-micron filter screen, washing the tissue agglomerates on the filter screen into a centrifuge tube by using a basic culture medium to reduce cell loss, and centrifuging for 3 minutes at room temperature of 1500 rpm;
3.5 abandoning the supernatant, observing whether blood cells exist, if the blood cells exist, adding 8mL of blood cell lysate (purchased from Sigma company), mixing uniformly, cracking at 4 ℃ for 20 minutes, reversing and mixing uniformly once, and centrifuging at 1500rpm at room temperature for 4 minutes;
3.6 discard the supernatant, add 2mL of basal medium to resuspend the cells for further use.
4 cell counting and processing
4.1 Observation under a lens: a small amount of the resuspended cells are removed and spread in a culture dish, and the density and morphology of the cancer cells are observed under a microscope (CNOPTEC, BDS 400);
4.2 viable cell count: after 12. Mu.L of the resuspended cell suspension and 12. Mu.L of trypan blue stain (manufacturer: biotechnology (Shanghai) Co., ltd.) were mixed well, 20. Mu.L of the mixture was added to a cell counting plate (manufacturer: countstar, specification: 50 plates/cassette), and the percentage of viable large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC 1000) = viable cell count/total cell count of 100%.
(3) Culture of cervical cancer organoids
Resuspending and counting the cervical cancer primary cells obtained in the step by using precooled DMEM/F12, and diluting the cell density to 5-10 multiplied by 10 5 mu.L/mL, 400. Mu.L of the diluted cell suspension was added to an equal volume of Matrigel (Corning) and gently mixed, and then the mixture was mixedThe material was inoculated at 8. Mu.L/well into a 96-well plate. The inoculated culture plate is placed into an incubator for 30 minutes, after the Matrigel is completely solidified, the culture media shown in table 1 which are restored to the room temperature in advance are respectively added, and the culture media are replaced once every five days for the expanded culture. The cultured organoids were photographed 10 days later, and the sizes of diameters of the organoids were measured and counted to compare the promoting effects of the factors on the proliferation of the cervical cancer organoids. Among them, as an experimental control, a basal medium without any additive was used, and the experimental results are shown in table 1.
TABLE 1 additional ingredients in culture Medium and organoid proliferation promoting Effect
Wherein "+" indicates that the culture medium added with the additive has the effect of promoting proliferation of at least two cases of cervical cancer organoids separated from cervical cancer tissues compared with a basal culture medium; "-" indicates that the medium to which the additive was added showed an inhibitory effect on proliferation of at least one of cervical cancer organoids isolated from cervical cancer tissues; ". Smallcircle" indicates that the medium to which the additive was added had no significant effect on the proliferation of at least two of the cervical cancer organoids isolated from cervical cancer tissues.
From the above results, factors such as compound 1, Y27632, SB202190, keratinocyte Growth Factor (KGF), hepatocyte Growth Factor (HGF), a83-01, B27, glutaMAX, fibroblast growth factor 10 (FGF 10), nicotinamide, and Epidermal Growth Factor (EGF) were selected and subjected to further culture experiments.
EXAMPLE 2 proliferation of cervical cancer organoids by Medium supplements at different concentrations
Cervical cancer primary cells were obtained from intraoperative tissue samples (nos. OC1, OC 2) according to the method of (2) of example 1, and organoid culture was performed using the medium formulation in table 2 below.
TABLE 2 culture Medium formulation (final concentration)
When the culture medium of formula 1 is used, 200 μ L of prepared B27 per well is added to a 96-well plate inoculated with organoids on the basis of formula 1, and the final concentrations of B27 are 1; and a control well (BC) was set using the medium of formulation 1. The final concentrations of other additional factors in the series of media were the same as COM media. The following experiments for formulations 1 to 11 were also performed in the same manner and will not be described in detail.
When the culture medium of the formula 2 is used, 200 mu L of prepared HGF is added into a 96-well plate inoculated with organoids on the basis of the formula 2, and the final concentrations of HGF are respectively 40ng/mL, 10ng/mL and 5ng/mL; and control wells (BC) were set using medium of formula 2.
When the culture medium of the formula 3 is used, 200 mu L of the prepared SB202190 cell culture additive is added to a 96-well plate inoculated with an organoid on the basis of the formula 3, wherein the final concentrations of the SB202190 cell culture additive are 200nM, 500nM and 1000nM respectively; and control wells (BC) were set using medium of formula 3.
When the culture medium of formula 4 is used, 200 μ L of prepared Y27632 per well is added to a 96-well plate inoculated with organoids on the basis of formula 4, and the final concentrations of Y27632 are 2.5 μ M, 5 μ M and 10 μ M respectively; and control wells (BC) were set using medium of formula 4.
When the culture medium of the formula 5 is used, 200 mu L of the prepared A83-01 is added into a 96-well plate inoculated with an organoid on the basis of the formula 5, wherein the final concentration of the A83-01 is 200nM, 500nM and 1000nM respectively; and control wells (BC) were set using medium of formula 5.
When the culture medium of the formula 6 is used, 200 mu L of the prepared EGF is added to a 96-well plate inoculated with an organoid on the basis of the formula 6, wherein the final concentration of the EGF is 1ng/mL, 5ng/mL and 40ng/mL respectively; and control wells (BC) were set using medium of formula 6.
When the culture medium of the formula 7 is used, 200 mu L of prepared FGF10 per well is added on the basis of the formula 7 in a 96-well plate inoculated with organoids, and the final concentration of the FGF10 is 10ng/mL, 20ng/mL and 100ng/mL respectively; and control wells (BC) were set using medium of formula 7.
When the culture medium of the formula 8 is used, 200 mu L of prepared KGF is respectively added into a 96-well plate inoculated with an organoid on the basis of the formula 8, and the final concentration of the KGF is respectively 2ng/mL, 10ng/mL and 40ng/mL; and control wells (BC) were set using medium of formula 8.
When the culture medium of formula 9 is used, 200 μ L of prepared GlutaMAX per well is added to a 96-well plate inoculated with organoids on the basis of formula 9, and the final concentration of GlutaMAX is 1; and control wells (BC) were set using medium of formulation 9.
When the culture medium of the formula 10 is used, 200 μ L of the prepared compound 1 per well is added to a 96-well plate inoculated with an organoid on the basis of the formula 10, and the final concentrations of the compound 1 are 2.5 μ M, 5 μ M and 10 μ M respectively; and control wells (BC) were set using medium of formula 10.
When the culture medium of the formula 11 is used, 200 mu L of prepared nicotinamide is added to a 96-well plate inoculated with the organoid on the basis of the formula 11, wherein the final concentration of the nicotinamide is 1mM, 2.5mM and 10mM respectively; and control wells (BC) were set using medium of formulation 11.
After 12 days, the cultured organoids were photographed, and the sizes of diameters of the organoids were measured and counted, and the effects of the concentrations of the factors on the promotion of the proliferation of the cervical cancer organoids were compared. The data collected for 2 samples are summarized in FIGS. 1A to 1K. In FIGS. 1A-1K, the ratio is the ratio of the organoid diameter obtained by 12 days of culture using each medium to the organoid diameter obtained by 12 days of culture in the corresponding BC control wells. The ratio is more than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is better than that of a control Kong Peiyang medium; if the ratio is less than 1, the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is weaker than that of the culture medium of the control hole.
According to the results of fig. 1A to 1K, the volume concentration of B27 is preferably 1; the preferred content of the hepatocyte growth factor HGF is 5-40 ng/mL; the content of SB202190 is preferably 200-1000 nM; the content of Y27632 is preferably 2.5-10 mu M; the content of A83-01 is preferably 200-1000 nM; the content of the epidermal growth factor EGF is preferably 1-40 ng/mL; the content of the fibroblast growth factor 10 is preferably 10-100 ng/mL; the content of the keratinocyte growth factor is preferably 2-40 ng/mL; the volume concentration of GlutaMAX is preferably 1; the content of the MST1/2 kinase inhibitor compound 1 is preferably 2.5-10 mu M; the nicotinamide content is preferably 1 to 10mM.
Example 3 cervical cancer organoid culture and characterization
The cervical cancer primary cells (OC 1, OC2 and OC 4) obtained according to the method of (2) of example 1 were resuspended and counted using the cervical cancer organoid culture medium COM of the present invention, and the cell density was diluted to 5 to 10X 10 5 mu.L/mL of the diluted cell suspension was taken out and added to an equal volume of Matrigel (Corning) and gently mixed, and then the mixture was seeded into a 24-well plate at 40. Mu.L/well. And (3) putting the inoculated culture plate into an incubator for 30 minutes until Matrigel is completely solidified, then adding a cervical cancer organoid culture medium COM which is restored to room temperature in advance, wherein each well is 500 mu L, and replacing the culture medium once every five days for amplification culture.
The cultured cervical cancer organoids were observed on days 5 to 14 using a microscope (EVOS M500, invitrogen). FIGS. 2A-2D are photographs of cervical cancer organoids taken under 4-fold objective lens after culturing of samples OC1 (day 5), OC1 (day 14), OC2 (day 7), and OC2 (day 7, another field). As shown, organoids continue to increase in volume during culture; the same sample can form different types of organoids and can simulate the heterogeneity of tumors in vitro.
And (3) performing pathological and immunohistochemical identification on the cervical cancer organoids obtained by culture, and performing pathological and immunohistochemical identification on corresponding original tissue samples at the same time, and comparing the consistency of the organoids and the histopathological indexes.
Fig. 3 shows the results of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoids obtained after in vitro culture, which are pictures photographed under a 20-fold objective lens, respectively. As shown in the figure, the results show that the structural morphology of organoids is cancer tissue morphology; the cells obtained after organoid culture of the sample in this example were judged to be cervical cancer cells according to immunohistochemical indicators. This result indicates that the cervical cancer organoids cultured with the culture medium COM of the present invention were consistent with the diagnosis results of the cervical cancer tissues before culture.
Example 4 comparison of the culture Effect of the prior art Medium
(1) Preparation of literature culture Medium
The formulation of the medium used in the formulation literature (Lohmussaar et al, 2021, cell Stem Cell 28, 1380-1396) was Advanced DMEM/F12 medium (from Invitrogen) +1: penicillin/streptomycin (from Corning Inc.) + 50. Mu.g/mL Primocin (from Invivogen Inc.) +1: 100. Mu.g/mL Primox (from Corning Inc.) +10mM HEPES (from Saymid. Co.) +1:50B27 (from Gibco Inc.) +1.25 mmol/LN-acetylcysteine (from MCE Inc.) +5mmol/L nicotinamide (from MCE Inc.) +500ng/mL R-Spondin 1 (from silicon biol Co. +25ng/mL keratinocyte growth factor (from silicon biol Co. +25 ng/mL) (from MCno biol. + 3262 mmol) + FGF + 10. Mu.g/mL FGF + 10. Mu.10. Mu.L (from MCo biol. + 3220. Mu.m biol. + 10. Mu.L biol Biochemical company) + (from MCE company) + Biol (from MCE +/3220. Mu.10 mL) and 3220. Mu.L biol (from MCe biol. + 3220. Mu.g/mL). Hereinafter referred to as ROM medium.
(2) Cervical cancer organoid culture
Cervical cancer primary cells were obtained from intraoperative tissue sample OC6 according to the method of (2) of example 1, and organoid culture was performed according to the method of example 3 using COM medium and ROM medium, respectively.
On the 10 th day of culture, the cultured cervical cancer organoids were observed using a microscope (EVOS M500, invitrogen). FIGS. 4A and 4B are photographs of organoids taken under a 4-fold objective lens and cultured in COM medium and ROM medium, respectively, for 10 days.
As can be seen from the results of fig. 4A and 4B, COM medium significantly promoted the formation and expansion culture of cervical cancer organoids as compared to ROM medium.
EXAMPLE 5 cervical cancer organoids amplified using the culture Medium of the present invention for drug screening
(1) Cervical cancer organoid culture
Cervical cancer primary cells were isolated from a cervical cancer intraoperative specimen (CCa 5) by the method of (2) of example 1, and organoid culture was performed using COM medium, and drug screening was performed when the diameter of the cervical cancer organoid exceeded 50 μm.
(2) Screening drug formulations
4 drugs (cisplatin, carboplatin, paclitaxel, and bortezomib; all from MCE) were formulated in 6 concentration gradients according to the following table and stored until use.
Preparation of cisplatin additive solutions with different concentrations: cisplatin was prepared into 6 additive solutions of different concentrations, the highest concentration was 9.5. Mu.M, and then diluted 2-fold to obtain additive solutions of different concentrations of 4.75. Mu.M, 2.38. Mu.M, 1.19. Mu.M, 0.59. Mu.M, and 0.3. Mu.M.
Preparing carboplatin additive solutions with different concentrations: carboplatin is prepared into 6 stock solutions with different concentrations, the highest concentration is 137.92 MuM, and then the stock solutions are diluted by 2 times of dilution ratio to obtain additive solutions with different concentrations of 68.96 MuM, 34.48 MuM, 17.24 MuM, 8.62 MuM and 4.31 MuM.
Preparing paclitaxel addition solutions with different concentrations: paclitaxel is prepared into 6 additive solutions with different concentrations, the highest concentration is 11.51 mu M, and then diluted by 2 times of dilution ratio to obtain additive solutions with different concentrations of 5.76 mu M, 2.88 mu M, 1.44 mu M, 0.72 mu M and 0.36 mu M.
Preparing bortezomib additive solutions with different concentrations: the bortezomib is prepared into 6 additive solutions with different concentrations, the highest concentration is 2 mu M, and then the additive solutions are diluted by 2 times of dilution ratio to obtain the additive solutions with different concentrations of 1 mu M, 0.5 mu M, 0.25 mu M, 0.125 mu M and 0.0625 mu M.
(3) Dosing
Taking out the prepared drug additive solution, and placing at room temperature. The organoids obtained by culturing according to step (1) were taken out from the incubator, the culture medium in the culture wells was removed, and additives containing drugs at different concentrations were gradually introduced into a 96-well transparent culture plate along the walls of the wells at a rate of 100. Mu.L per well. After the dosing is finished, the surface of the 96-well plate is disinfected and then is transferred to an incubator to be cultured continuously, and the organoid activity is measured after 5 days.
(4) Organoid viability assay
Taking out CellTiter-Glo luminescent reagent (purchased from Promega) from a refrigerator at 4 ℃, putting 10ml of reagent into a sample adding groove, taking out a 96-well plate to be detected from an incubator, adding 50 mu LCellTiter-Glo luminescent reagent into each well, standing for 30 minutes, observing the state of cells in the 96-well plate, if most of the cells are cracked, shaking gently and mixing uniformly, absorbing 100 mu L of the mixture into the other white 96-well plate, and detecting by using a multifunctional enzyme-linked immunosorbent (Envision of Perkin Elmer).
(5) Data processing
The chemical luminescence value of the culture well on the fifth day is determined according to the formula, wherein the drug inhibition ratio (%) =100% - (day five) Drug treatment group Day zero culture well chemiluminescence values Drug treatment group ) /(day five culture well chemiluminescence values DMSO Chemiluminescence values of culture wells on day zero DMSO ) 100%, inhibition rates of different drugs were calculated and the results are shown in figure 5. FIG. 5 is a plot of the inhibition rate of test drugs at different concentrations for inhibiting cervical cancer organoid growth. Among the four antitumor drugs, bortezomib has a strong effect of inhibiting organoid growth under 6 concentrations, the inhibition efficiency of paclitaxel is kept consistent under 6 concentrations, and the inhibition effects of cisplatin and carboplatin with different concentrations have certain difference and are dose-dependent, which indicates that organoids of the same patient have different effectiveness and sensitivity to different drugs. According to the results, the effectiveness and the effective dosage of the medicine in clinical use of cervical cancer patients can be judged.
Industrial applicability
The invention provides a culture medium and a culture method for cervical cancer organoid culture, which can be used for evaluating and screening the curative effect of medicaments by using the organoids obtained by culture. Thus, the present invention is suitable for industrial applications.
Although the present invention has been described in detail herein, the present invention is not limited thereto, and modifications can be made by those skilled in the art based on the principle of the present invention, and thus, it is to be understood that various modifications made in accordance with the principle of the present invention are within the scope of the present invention.
Claims (10)
1. A culture medium for a cervical cancer organoid comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632, A83-01, epidermal growth factor, fibroblast growth factor 10, keratinocyte growth factor, glutaMAX and nicotinamide,
wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,
wherein the content of the first and second substances,
R 1 selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R 6 Substituted aryl, arylC 1-C6 alkyl and heteroaryl;
R 2 and R 3 Each independently selected from C1-C6 alkyl;
R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl;
R 6 selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
2. The culture medium of claim 1, wherein
R 1 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R 6 Substituted phenyl, naphthyl, benzyl and thienyl;
R 2 and R 3 Each independently selected from C1-C3 alkyl;
R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, piperidinylC 1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;
R 6 selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
3. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
wherein the content of the first and second substances,
R 1 selected from C1-C6 alkyl, optionally substituted by 1-2 independent R 6 Substituted phenyl, optionally substituted with 1-2 independent R 6 Substituted thienyl, and optionally substituted with 1-2 independent R 6 A substituted benzyl group;
R 5 selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;
R 6 each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.
4. The culture medium of claim 3, wherein
R 1 Is optionally substituted by 1-2 independent R 6 Substituted phenyl;
R 5 is hydrogen;
R 6 preferably fluorine, methyl or trifluoromethyl.
5. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or a pharmaceutically acceptable salt thereof:
6. a culture medium according to any one of claims 1 to 5, wherein the content of each component in the culture medium satisfies any one or more or all of the following:
the concentration of the MST1/2 kinase inhibitor is 2.5-10 mu M;
the volume ratio of the B27 or N2 cell culture additive to the culture medium is 1;
the concentration of the hepatocyte growth factor is 5-40 ng/mL;
the concentration of the SB202190 is 200-1000 nM;
the concentration of the Y27632 is 2.5-10 mu M;
the concentration of the A83-01 is 200-1000 nM;
the concentration of the epidermal cell growth factor is 1-40 ng/mL;
the concentration of the fibroblast growth factor 10 is 10-100 ng/mL;
the concentration of the keratinocyte growth factor is 2-40 ng/mL;
the volume ratio of the GlutaMAX to the culture medium is 1;
the concentration of the nicotinamide is 1-10 mM.
7. The culture medium according to any one of claims 1 to 5, further comprising:
a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and
an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin.
8. The culture medium according to any one of claims 1 to 5, wherein the culture medium is free of Wnt agonists, R-spondin family proteins, noggin proteins, and BMP inhibitors.
9. A method for culturing a cervical cancer organoid, characterized by comprising the steps of:
(1) Separating a sample from a cervical cancer solid tumor tissue to obtain cervical cancer primary cells;
(2) Preparing a culture medium for a cervical cancer organoid according to any one of claims 1 to 8, and organoid culturing the cervical cancer primary cells obtained in step (1).
10. A method for evaluating or screening a drug for treating cervical cancer, comprising the steps of:
(1) Culturing the cervical cancer organoids using the cervical cancer organoid culturing method of claim 9;
(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;
(3) Adding the diluted medicine to the cervical cancer organoid cultured in the step (1); and
(4) Organoid size or organoid viability detection is performed.
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| CN116555165A (en) * | 2023-06-29 | 2023-08-08 | 重庆大学附属肿瘤医院 | Cervical cancer organoid culture medium and application thereof |
| CN116716236A (en) * | 2023-06-29 | 2023-09-08 | 重庆大学附属肿瘤医院 | Construction method and application of cervical cancer organoids |
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| CN110734894B (en) * | 2019-10-11 | 2021-09-24 | 纳肽得(青岛)生物医药有限公司 | Universal cancer organoid in vitro culture medium |
| CN113528444B (en) * | 2020-04-15 | 2023-05-23 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium for esophageal squamous carcinoma epithelial cells, culture method and application thereof |
| CN113122500B (en) * | 2021-03-18 | 2022-08-02 | 上海诺典生物科技有限公司 | Culture and application of metastatic intestinal cancer organoid |
| CN113308437B (en) * | 2021-05-28 | 2023-03-14 | 丹望医疗科技(上海)有限公司 | Culture medium, method and kit for rapidly culturing bone metastasis cancer organoids |
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| CN116716236A (en) * | 2023-06-29 | 2023-09-08 | 重庆大学附属肿瘤医院 | Construction method and application of cervical cancer organoids |
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