CN111411082A - Culture medium for culturing CD90posi cells and culture method thereof - Google Patents
Culture medium for culturing CD90posi cells and culture method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of cell culture, and particularly relates to a culture medium for culturing CD90posi cells and a culture method thereof. The culture medium of CD90posi cells provided by the invention is characterized in that a PD-1 inhibitor and matrine are added into a basal culture medium DMEM/F-12 according to the ratio of 7: 3, the cell growth inhibitor is prepared from matsutake mushroom extract and acanthopanax extract according to the weight ratio of 3: 2 weight ratio of mutation inhibitor; the CD90posi cell is cultured by adopting a three-dimensional culture method. The culture medium provided by the invention is adopted to culture the CD90posi cells, and has the following advantages: the cells are not easy to aggregate, the culture speed is easy to control, simultaneously, the activity of the cells in the culture process can be ensured, and the problem that the cells continuously mutate in the culture process can not occur.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a culture medium for culturing CD90posi cells and a culture method thereof.
Background
The primary liver cancer is one of the most common malignant tumors in China, has high morbidity and high mortality, is the second place of domestic tumors in the disease mortality, and seriously threatens the life health of people. The liver cancer is hidden, the clinical manifestations lack specificity, the liver cancer is usually in the middle and late stage of the liver cancer when hospitalized, patients in the early stage of the liver cancer are difficult to find, and the treatment effect of the liver cancer is seriously influenced.
Currently, the clinical detection means of liver cancer mainly detects serum alpha-fetoprotein (AFP) of a patient and combines with imaging examination to make a definite diagnosis, and the clinical detection means can be divided into the following categories: the B-ultrasonic examination is influenced by intestinal gas and peripheral organs, the resolution is low, and a certain false negative rate exists when liver focus puncture biopsy is carried out under the guidance of the B-ultrasonic examination; meanwhile, serious complications such as wound bleeding and needle transfer exist in liver puncture. CT or MR can only distinguish focus with diameter over 1.0cm, and has certain false positive rate and false negative rate. While the DSA technique can improve the accuracy of liver cancer detection, it is an invasive test and is often used for liver cancer treatment. Because the AFP (alpha-fetoprotein) in serum is remarkably increased and generally appears in the advanced stage of tumor, the positive rate is only 60-70%, the method has obvious limitations in the aspects of accuracy, detection efficiency, treatment risk of patients after diagnosis disturbance, delay of diagnosis time and the like, and the development of a new detection means has importance and urgency.
Chinese patent application CN108872603A discloses an identification method for liver cancer stem cells, which can ensure the stable expression of marker proteins, and the expression level and the L CSC cell content present a good linear relationship, so as to realize the relative quantitative analysis of L CSC cell content, but does not solve the process of how to obtain a large amount of liver cancer cells in the prior art, and can better analyze the liver cancer cells only by realizing the in vitro large amount of proliferation of the liver cancer cells.
In conclusion, the prior art lacks an effective method for culturing liver cancer cells, and the liver cancer cells cultured by the prior culture medium have the defects of easy aggregation, difficult control, easy mutation and the like.
In the analysis of hepatocellular carcinoma microenvironment cell population, we screened out CD90posi tumor cells, and found that CD90posi cells promote proliferation and migration of liver cancer cells. Meanwhile, the CD90posi cell shows strong proliferation potential and cell dryness. The CD90posi hepatoma cells are injected into nude mice subcutaneously, and the transplanted tumor volume and weight are larger along with the increase of the proportion of the CD90posi cells, which indicates that the CD90posi cells are a key factor for proliferation and growth of hepatocellular carcinoma.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a culture medium for culturing CD90posi cells and a culture method thereof. The CD90posi cells cultured by the culture medium provided by the invention are not easy to aggregate, the culture speed is easy to control, simultaneously, the activity of the cells in the culture process can be ensured, and the problem that the cells continuously mutate in the culture process can not occur.
In order to achieve the purpose, the invention adopts the technical scheme that:
a culture medium for culturing CD90posi cells comprises the following components in percentage by weight: 94-97.5% of DMEM/F-12 basal medium, 0.5-3% of cell growth inhibitor and 2-3% of mutation inhibitor.
Preferably, the culture medium for culturing the CD90posi cells consists of the following components in percentage by weight: 96% of DMEM/F-12 basal medium, 1.5% of cell growth inhibitor and 2.5% of mutation inhibitor.
Preferably, the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 6-8: 2-4; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 2-4: 1 to 3.
Preferably, the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 7: 3, preparing a composition; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 3: 2.
The invention also provides a method for culturing CD90posi cells by using the culture medium, which comprises the following steps:
s1, cutting fresh liver cancer tissues under aseptic conditions, culturing for two generations, and separating CD90posi cells by an immunomagnetic bead indirect labeling separation method to obtain CD90posi cells;
s2, digesting the CD90posi cells obtained in the step S1 with matrigel mother liquor, then re-suspending the cells in the culture medium, treating the cells on ice in the whole process, and collecting the cell suspension to obtain a precooled cell suspension after digestion of the matrigel mother liquor;
s3, digesting the matrigel mother liquor obtained in the step S2, and then pre-cooling the cell suspension and the matrigel solution according to the volume ratio of 1-2: 3-5, uniformly mixing, placing at 34-37 ℃, and standing for 35-45 min to form gel; obtaining a colloidal mixed culture;
s4, adding the culture medium into the colloidal mixed culture obtained in the step S3, and placing at 36 ℃ and 3% CO2And (5) incubating in an incubator, changing the liquid once in 24 hours, and culturing to obtain the compound feed.
Preferably, the specific process of separating CD90posi cells by the immunomagnetic bead indirect labeling separation method in step S1 is as follows:
(1) taking out frozen liver cancer tissue from a refrigerator at the temperature of-80 ℃, rewarming at the temperature of 37 ℃, washing with PBS, centrifuging for 5min at the rotating speed of 1600rpm, collecting supernatant, repeatedly washing for 2 times, adding 15m L DMEM/F12+ 10% PBS culture solution, gently blowing, resuspending cells to uniformly disperse, transferring into a 100m L culture bottle, culturing at the temperature of 37 ℃ with 5% CO2Culturing under environment, and subculturing when the cells are spread to 80% of the bottom of the bottle to obtain primary culture cells;
(2) digesting the primary culture cells obtained in the step (1) by using 0.25% trypsin, blowing down adherent cells by using a rubber head pipette after the cells are observed to be separated from the bottom of a bottle under an inverted microscope, adding a proper amount of FBS (bovine serum albumin) to terminate digestion, transferring the cells into a centrifuge tube, adding PBS (phosphate buffer solution) to remove residual enzyme, centrifuging the cells for 5min at the rotating speed of 1600rpm, removing supernatant, re-suspending the cells by using 1m L culture solution, fully and uniformly mixing the cells, adding the cells into the culture bottle, continuously adding 5m L culture solution, and performing 5% CO treatment at 37 ℃ and 5%2Culturing under environment to obtain subculture cells;
(3) digesting the primary culture cells obtained in the step (2) by using 0.25% trypsin, fully blowing and beating to prepare a single cell suspension, centrifuging for 5min at the rotating speed of 1600rpm, removing a supernatant, resuspending the cells by using 10m L phosphate buffer solution, then sucking the cell suspension by using a needle tube, quickly pushing out to disperse the cells cohered into a cluster, adding 1m L of Anti-Human CD90 PE antibody, uniformly mixing, incubating for 30min at 4 ℃ in the dark, taking out and shaking uniformly every 10min, washing twice by using phosphate buffer solution, removing the cells which are not combined with one antibody, centrifuging for 5min at the rotating speed of 1600rpm, removing the supernatant, adding 2m L of Anti-PEMicrobed antibody, incubating for 30min at 4 ℃ in the dark, washing twice by using phosphate buffer solution, removing the cells which are not combined with a secondary antibody, adding 1m L of phosphate buffer solution, placing an MS sorting column into a magnetic field fixed on a sorter, wetting the column by using 1m L of phosphate buffer solution, then adding a pipette gun into the cell sorting column, slowly adding L min of magnetic bead into the magnetic sorting column, removing the cell suspension carried by using 355631 m magnetic bead suspension, adding the cell sorting column, removing the cell suspension, and washing the cell sorting column, removing the cell suspension under the magnetic bead 3526 cell suspension, and washing by using a magnetic piston 355631 m magnetic cell sorting column, and washing piston, and washing to obtain a centrifugal tube, and washing cell sorting column, and separating the cell sorting column, and adding the cell suspension under the cell sorting.
Preferably, the mass concentration of the matrigel mother liquor in the step S2 is 9-10 mg/m L.
Preferably, the mass concentration of the matrigel solution in the step S3 is 10-11 mg/m L.
In the invention, when culturing the CD90posi cells, a cell growth inhibitor which is composed of a PD-1 inhibitor and matrine according to a certain proportion is added into a culture medium, so that the unlimited growth of cancer cells can be effectively inhibited, and the in-vitro culture speed of the cancer cells can be controlled; meanwhile, a mutation inhibitor which is composed of a tricholoma matsutake extract and an acanthopanax senticosus extract according to a certain proportion is added in the application to prevent CD90posi cells from mutating in the culture process so as to improve the cell culture purity; in the process of culturing the CD90posi cells, a three-dimensional culture method is adopted, so that the three-dimensional culture method is closer to the in-vivo environment, the proliferation and differentiation capacity of cancer cells is kept, the simulation of the in-vivo environment by people is facilitated, and the further deep research on the liver cancer cells is realized.
In addition, the applicant also unexpectedly finds that the problem of aggregation and agglomeration of the CD90posi cells can be effectively avoided by adding the cell growth inhibitor and the mutation inhibitor into the culture medium at the same time, so that the cultured cells are easily separated into a monolayer, and the cells are conveniently counted and the liver cancer is conveniently researched.
Compared with the prior art, the culture medium for culturing the CD90posi cells and the culture method thereof provided by the invention have the following advantages:
(1) the culture medium for culturing the CD90posi cells can control the cell culture speed and prevent the cells from generating mutation in the culture process;
(2) the culture medium for culturing the CD90posi cells can effectively avoid the problem of aggregation and conglobation of the CD90posi cells, so that the cultured cells are easy to separate into a monolayer;
(3) when the culture medium provided by the invention is used for culturing the CD90posi cells, a three-dimensional culture method is adopted, so that the three-dimensional culture method is closer to the internal environment of the cells, the activity of cancer cells can be effectively maintained, and the subsequent deep research on liver cancer is facilitated.
Drawings
FIG. 1 shows the aggregation of CD90posi cells cultured in the example 3 group and the comparative example 3 group.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The PD-1 inhibitor is available from beijing baiolai bock technologies ltd; the matrine can be purchased from Shanghai Hui Shi medicine science and technology Limited; the Tricholoma matsutake extract and the acanthopanax senticosus extract can be purchased from Xianminglang biological technology limited; the Anti-Human CD90 PE antibody and the Anti-PE Microbed antibody can be purchased from of Shanghai, Yongda industries, Co., Ltd; the other reagents are common reagents and can be purchased from conventional reagent production and sale companies.
EXAMPLE 1A Medium for culturing CD90posi cells
The culture medium for culturing the CD90posi cells comprises the following components in percentage by weight: 97.5 percent of DMEM/F-12 basal medium, 0.5 percent of cell growth inhibitor and 2 percent of mutation inhibitor; the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 6: 2, preparing a composition; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 2: 1.
A method for culturing CD90posi cells by using the culture medium comprises the following steps:
s1, cutting fresh liver cancer tissues under aseptic conditions, culturing for two generations, and separating CD90posi cells by an immunomagnetic bead indirect labeling separation method to obtain CD90posi cells; the specific process for separating the CD90posi cells by the immunomagnetic bead indirect labeling separation method comprises the following steps:
(1) taking out frozen liver cancer tissue from a refrigerator at the temperature of-80 ℃, rewarming at the temperature of 37 ℃, washing with PBS, centrifuging for 5min at the rotating speed of 1600rpm, collecting supernatant, repeatedly washing for 2 times, adding 15m L DMEM/F12+ 10% PBS culture solution, gently blowing, resuspending cells to uniformly disperse, transferring into a 100m L culture bottle, culturing at the temperature of 37 ℃ with 5% CO2Culturing under environment, and subculturing when the cells are spread to 80% of the bottom of the bottle to obtain primary culture cells;
(2) digesting the primary culture cells obtained in the step (1) by using 0.25% trypsin, blowing down adherent cells by using a rubber head pipette after the cells are observed to be separated from the bottom of a bottle under an inverted microscope, adding a proper amount of FBS (bovine serum albumin) to terminate digestion, transferring the cells into a centrifuge tube, adding PBS (phosphate buffer solution) to remove residual enzyme, centrifuging the cells for 5min at the rotating speed of 1600rpm, removing supernatant, re-suspending the cells by using 1m L culture solution, fully and uniformly mixing the cells, adding the cells into the culture bottle, continuously adding 5m L culture solution, and performing 5% CO treatment at 37 ℃ and 5%2Culturing under environment to obtain subculture cells;
(3) digesting the primary culture cells obtained in the step (2) by using 0.25% trypsin, fully blowing and beating to prepare a single cell suspension, centrifuging for 5min at the rotating speed of 1600rpm, removing a supernatant, resuspending the cells by using 10m L phosphate buffer, then sucking the cell suspension by using a needle tube, quickly pushing out to disperse the cells cohered into a cluster, adding 1m L of Anti-Human CD90 PE antibody, uniformly mixing, incubating for 30min at 4 ℃ in the dark, taking out and shaking uniformly every 10min, washing twice by using phosphate buffer, removing the cells which are not combined with one antibody, centrifuging for 5min at the rotating speed of 1600rpm, removing the supernatant, adding 2m L of Anti-PEMicrobed antibody, incubating for 30min at 4 ℃ in the dark, washing twice by using phosphate buffer, removing the cells which are not combined with a secondary antibody, adding 1m L of phosphate buffer, placing an MS sorting column into a magnetic field fixed on a sorter, wetting the column by using 1m of phosphate buffer of L m L, then adding a pipette gun into the cell sorting column, slowly adding the cell sorting column, taking the cell suspension carried by a magnetic bead, carrying the cell sorting column, removing L cell suspension, adding 355631 m magnetic cell sorting buffer into the cell suspension, and washing the cell suspension, removing the cell suspension under the magnetic cell sorting column, and washing by using a piston 355631 m magnetic cell sorting column, and washing piston 3526 magnetic cell sorting cylinder, and washing to obtain a centrifugal tube, and washing cell sorting column, and separating the cell sorting column;
s2, digesting the CD90posi cells obtained in the step S1 with matrigel mother liquor, then re-suspending the cells in the culture medium, treating the cells on ice in the whole process, and collecting the cell suspension to obtain a precooled cell suspension after digestion of the matrigel mother liquor, wherein the mass concentration of the matrigel mother liquor is 9mg/m L;
s3, uniformly mixing the cell suspension obtained by digesting the matrigel mother liquor and precooled in the step S2 with the matrigel solution according to the volume ratio of 1: 3, placing at 34 ℃ for 35min to gelatinize to obtain a gelatinous mixed culture, wherein the mass concentration of the matrigel solution is 10mg/m L;
s4, adding the culture medium into the colloidal mixed culture obtained in the step S3, and placing at 36 ℃ and 3% CO2And (5) incubating in an incubator, changing the liquid once in 24 hours, and culturing to obtain the compound feed.
Example 2A Medium for culturing CD90posi cells
The culture medium for culturing the CD90posi cells comprises the following components in percentage by weight: 94% of DMEM/F-12 basal medium, 3% of cell growth inhibitor and 3% of mutation inhibitor; the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 8: 4, preparing a composition; the mutation inhibitor is prepared from matsutake mushroom extract and acanthopanax extract according to a weight ratio of 4: 3, preparing a composition;
a method for culturing CD90posi cells by using the culture medium comprises the following steps:
s1, cutting fresh liver cancer tissues under aseptic conditions, culturing for two generations, and separating CD90posi cells by an immunomagnetic bead indirect labeling separation method to obtain CD90posi cells; the specific process for separating the CD90posi cells by the immunomagnetic bead indirect labeling separation method is the same as the embodiment;
s2, digesting the CD90posi cells obtained in the step S1 by matrigel mother liquor, then suspending the cells in the culture medium, placing the cells on ice for treatment in the whole process, and collecting the cell suspension to obtain precooled cell suspension after digestion of the matrigel mother liquor, wherein the mass concentration of the matrigel mother liquor is 10mg/m L;
s3, uniformly mixing the cell suspension obtained by digesting the matrigel mother liquor and precooled in the step S2 with the matrigel solution according to the volume ratio of 2: 5, placing at 37 ℃ for 45min to gelatinize to obtain a gelatinous mixed culture, wherein the mass concentration of the matrigel solution is 11mg/m L;
s4, adding the culture medium into the colloidal mixed culture obtained in the step S3, and placing at 36 ℃ and 3% CO2And (5) incubating in an incubator, changing the liquid once in 24 hours, and culturing to obtain the compound feed.
EXAMPLE 3A Medium for culturing CD90posi cells
The culture medium for culturing the CD90posi cells comprises the following components in percentage by weight: 96% of DMEM/F-12 basal medium, 1.5% of cell growth inhibitor and 2.5% of mutation inhibitor; the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 7: 3, preparing a composition; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 3: 2.
A method for culturing CD90posi cells by using the culture medium comprises the following steps:
s1, cutting fresh liver cancer tissues under aseptic conditions, culturing for two generations, and separating CD90posi cells by an immunomagnetic bead indirect labeling separation method to obtain CD90posi cells; the specific process for separating the CD90posi cells by the immunomagnetic bead indirect labeling separation method is the same as the embodiment;
s2, digesting the CD90posi cells obtained in the step S1 with matrigel mother liquor, then re-suspending the cells in the culture medium, treating the cells on ice in the whole process, and collecting the cell suspension to obtain a precooled cell suspension after digestion of the matrigel mother liquor, wherein the mass concentration of the matrigel mother liquor is 9.5mg/m L;
s3, uniformly mixing the cell suspension obtained by digesting the matrigel mother liquor and precooled in the step S2 with the matrigel solution according to the volume ratio of 1.5: 4, placing at 36 ℃, standing for 40min, and gelatinizing to obtain a gelatinous mixed culture, wherein the mass concentration of the matrigel solution is 10.5mg/m L;
s4, adding the culture medium into the colloidal mixed culture obtained in the step S3, and placing at 36 ℃ and 3% CO2And (5) incubating in an incubator, changing the liquid once in 24 hours, and culturing to obtain the compound feed.
Comparative example 1A Medium for culturing CD90posi cells
The culture medium for culturing the CD90posi cells comprises the following components in percentage by weight: 96% of DMEM/F-12 basal medium, 1.5% of cell growth inhibitor and 2.5% of mutation inhibitor; the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 1: 1, preparing a composition; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 3: 2.
The method for culturing the CD90posi cells by using the culture medium is similar to the method in the example 3;
the difference from example 3 is that the cell growth inhibitor in comparative example 1 is prepared from PD-1 inhibitor and matrine in a weight ratio of 1: 1.
Comparative example 2A Medium for culturing CD90posi cells
The culture medium for culturing the CD90posi cells comprises the following components in percentage by weight: 96% of DMEM/F-12 basal medium, 1.5% of cell growth inhibitor and 2.5% of mutation inhibitor; the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 7: 3, preparing a composition; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 1: 1.
The method for culturing the CD90posi cells by using the culture medium is similar to the method in the example 3;
the difference from example 3 is that the mutation inhibitor in comparative example 2 is prepared from matsutake mushroom extract and acanthopanax senticosus extract in a weight ratio of 1: 1.
Comparative example 3A Medium for culturing CD90posi cells
The culture medium for culturing the CD90posi cells comprises the following components in percentage by weight: 97.5 percent of DMEM/F-12 basal medium and 2.5 percent of mutation inhibitor; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 3: 2.
The method for culturing the CD90posi cells by using the culture medium is similar to the method in the example 3;
the difference from example 3 is that comparative example 3 does not contain a cytostatic agent.
Comparative example 4A Medium for culturing CD90posi cells
The culture medium for culturing the CD90posi cells comprises the following components in percentage by weight: 96% of DMEM/F-12 basal medium, 1.5% of cell growth inhibitor and 2.5% of mutation inhibitor; the cell growth inhibitor is prepared from a PD-1 inhibitor and matrine in a weight ratio of 7: 3, preparing a composition; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 3: 2.
The difference from example 3 is that comparative example 4 uses the medium to culture CD90posi cells using conventional subculture methods.
Test example 1 cell viability assay
1. Test samples: the culture media of examples 1-3 and comparative example 1, comparative example 4 and the method for culturing CD90posi cells;
2. test method, the cell suspension cultured in the groups of examples 1-3 and comparative example 1 and comparative example 4 is respectively diluted to 2 × 10m L4Adding 0.5m L into a test tube, adding 0.5m L, 4% trypan blue staining solution into the test tube, staining for 2-3 min, sucking a little suspension, coating the suspension on a glass slide, adding a cover plate, counting the number of dead cells and the number of live cells in any field of view under a microscope, and calculating the cell activity.
Cell viability-viable cell number/(dead cell number + viable cell number) × 100%
3. And (3) test results: the specific test results are shown in table 1.
TABLE 1 comparison of viability of different cell samples
| Test sample | Total number of cells/cell | Number of living cells/cell | Cell viability/% |
| EXAMPLE 1 group | 1500 | 1404 | 93.6 |
| EXAMPLE 2 group | 1500 | 1422 | 94.8 |
| EXAMPLE 3 group | 1500 | 1458 | 97.2 |
| Comparative example 1 group | 1500 | 1284 | 85.6 |
| Comparative example 4 group | 1500 | 1152 | 76.8 |
As can be seen from table 1, the cell viability of the cells cultured in the groups 1 to 3 of the present invention can reach 90% or more, especially the cell viability of the group 3 of the present invention reaches 97.2%, so the group 3 of the present invention is the best embodiment of the present invention; while the activity of the CD90posi cells cultured by the conventional subculture method is 76.8% in comparative example 4, it can be seen that the cell activity is greatly improved by culturing the CD90posi cells by the three-dimensional culture method, and the cell activity is also reduced by changing the culture medium in the group of comparative example 1.
Test example 2 Observation of cell aggregability
1. Test samples: example 3 and comparative example 3 groups of cultured cells
2. The test method comprises the steps of respectively taking 10 mu L of cell suspensions of the groups of example 3 and the group of comparative example 3, adding 90 mu L of normal saline for dilution, shaking and uniformly mixing, taking a small amount of the cell suspensions, placing the small amount of the cell suspensions on a glass slide, adding a cover plate, taking a clear visual field under a microscope, and observing the aggregation and agglomeration of cells.
3. And (3) test results: the specific test results are shown in FIG. 1.
In FIG. 1, a represents cells cultured in example 3 group of the present invention, and b represents cells cultured in comparative example 3 group; as can be seen from FIG. 1, the cells of the example 3 group of the present invention showed no significant aggregation, whereas the cells of the comparative example 3 group showed severe aggregation.
Test example 3 detection of cell mutation
1. Test samples: example 3 and comparative example 2 groups
2. The test method comprises diluting a small amount of cell suspension of example 3 and comparative example 2 by 100 times, mixing, dropping one drop on a glass slide, adding a cover plate, randomly selecting several visual fields under an electron microscope, observing the morphological change of cells and the change of chromosome and nucleolus structure, and calculating the cell mutation rate (total observed cell/observed cell number: × 100%)
3. And (3) test results: the specific test results are shown in Table 2.
TABLE 2 comparison of mutation results of cell samples cultured by different methods
| Test sample | Total number of cells/cell | Number of mutant cells/cell | Mutation rate/% |
| EXAMPLE 3 group | 500 | 3 | 0.6 |
| Comparative example 2 group | 500 | 87 | 17.4 |
As can be seen from Table 2, the cell mutation rate of CD90posi cultured in the medium of the inventive example 3 group was only 0.6%, whereas the cell mutation rate of the comparative example 2 group was as high as 17.4%, since the comparative example 2 group changed the ratio of each component in the cell mutation inhibitor.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A culture medium for culturing CD90posi cells is characterized by comprising the following components in percentage by weight: 94-97.5% of DMEM/F-12 basal medium, 0.5-3% of cell growth inhibitor and 2-3% of mutation inhibitor.
2. The medium for culturing CD90posi cells according to claim 1, consisting of the following components in percentage by weight: 96% of DMEM/F-12 basal medium, 1.5% of cell growth inhibitor and 2.5% of mutation inhibitor.
3. The culture medium for culturing CD90posi cells according to claim 1 or 2, wherein the cell growth inhibitor is prepared by mixing a PD-1 inhibitor and matrine in a weight ratio of 6-8: 2-4; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 2-4: 1 to 3.
4. The medium for culturing CD90posi cells according to claim 3, wherein the cytostatic agent is a mixture of PD-1 inhibitor and matrine in a weight ratio of 7: 3, preparing a composition; the mutation inhibitor is prepared from a tricholoma matsutake extract and an acanthopanax senticosus extract according to a weight ratio of 3: 2.
5. A method for culturing CD90posi cells using the medium of any one of claims 1 to 4, comprising the steps of:
s1, cutting fresh liver cancer tissues under aseptic conditions, culturing for two generations, and separating CD90posi cells by an immunomagnetic bead indirect labeling separation method to obtain CD90posi cells;
s2, digesting the CD90posi cells obtained in the step S1 with matrigel mother liquor, then re-suspending the cells in the culture medium of any one of claims 1 to 4, treating the cells on ice all the way, and collecting the cell suspension to obtain a cell suspension precooled after digestion of the matrigel mother liquor;
s3, digesting the matrigel mother liquor obtained in the step S2, and then pre-cooling the cell suspension and the matrigel solution according to the volume ratio of 1-2: 3-5, uniformly mixing, placing at 34-37 ℃, and standing for 35-45 min to form gel; obtaining a colloidal mixed culture;
s4, adding the culture medium of any one of claims 1-4 to the colloidal mixed culture obtained in step S3, and placing at 36 ℃ with 3% CO2Incubation in incubator, 24h liquid changeOnce, culturing to obtain the product.
6. The method for culturing CD90posi cells according to claim 5, wherein the specific process of separating CD90posi cells by immunomagnetic bead indirect labeling separation method in step S1 is as follows:
(1) taking out frozen liver cancer tissue from a refrigerator at the temperature of-80 ℃, rewarming at the temperature of 37 ℃, washing with PBS, centrifuging for 5min at the rotating speed of 1600rpm, collecting supernatant, repeatedly washing for 2 times, adding 15m L DMEM/F12+ 10% PBS culture solution, gently blowing, resuspending cells to uniformly disperse, transferring into a 100m L culture bottle, culturing at the temperature of 37 ℃ with 5% CO2Culturing under environment, and subculturing when the cells are spread to 80% of the bottom of the bottle to obtain primary culture cells;
(2) digesting the primary culture cells obtained in the step (1) by using 0.25% trypsin, blowing down adherent cells by using a rubber head pipette after the cells are observed to be separated from the bottom of a bottle under an inverted microscope, adding a proper amount of FBS (bovine serum albumin) to terminate digestion, transferring the cells into a centrifuge tube, adding PBS (phosphate buffer solution) to remove residual enzyme, centrifuging the cells for 5min at the rotating speed of 1600rpm, removing supernatant, re-suspending the cells by using 1m L culture solution, fully and uniformly mixing the cells, adding the cells into the culture bottle, continuously adding 5m L culture solution, and performing 5% CO treatment at 37 ℃ and 5%2Culturing under environment to obtain subculture cells;
(3) digesting the primary culture cells obtained in the step (2) by using 0.25% trypsin, fully blowing and beating to prepare a single cell suspension, centrifuging for 5min at the rotating speed of 1600rpm, removing a supernatant, resuspending the cells by using 10m L phosphate buffer solution, then sucking the cell suspension by using a needle tube, quickly pushing out to disperse the cells cohered into a cluster, adding 1m L of Anti-Human CD90 PE antibody, uniformly mixing, incubating for 30min at 4 ℃ in the dark, taking out and shaking uniformly every 10min, washing twice by using phosphate buffer solution, removing the cells which are not combined with one antibody, centrifuging for 5min at the rotating speed of 1600rpm, removing the supernatant, adding 2m L of Anti-PEMicrobed antibody, incubating for 30min at 4 ℃ in the dark, washing twice by using phosphate buffer solution, removing the cells which are not combined with a secondary antibody, adding 1m L of phosphate buffer solution, placing an MS sorting column into a magnetic field fixed on a sorter, wetting the column by using 1m L of phosphate buffer solution, then adding a pipette gun into the cell sorting column, slowly adding L min of magnetic bead into the magnetic sorting column, removing the cell suspension carried by using 355631 m magnetic bead suspension, adding the cell sorting column, removing the cell suspension, and washing the cell sorting column, removing the cell suspension under the magnetic bead 3526 cell suspension, and washing by using a magnetic piston 355631 m magnetic cell sorting column, and washing piston, and washing to obtain a centrifugal tube, and washing cell sorting column, and separating the cell sorting column, and adding the cell suspension under the cell sorting.
7. The method for culturing CD90posi cells according to claim 5, wherein the matrigel mother liquor of step S2 has a mass concentration of 9-10 mg/m L.
8. The method for culturing CD90posi cells according to claim 5, wherein the matrigel solution of step S3 has a mass concentration of 10-11 mg/m L.
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