CN110295145B - Culture solution and culture method for in vitro culture of brain tumor cells - Google Patents
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Abstract
The invention provides a culture solution for culturing brain tumor cells in vitro, which comprises a nerve cell basic culture medium, 0.5-5% of B27 trophic factor, 0.5-5% of double-resistant-penicillin/streptomycin, 0.5-5% of glutamine, 0.5-5% of sodium pyruvate, 0.5-1.5% of fetal calf serum and 2.5-3.5 ug/mL recombinant Shh protein based on the mass of the nerve cell basic culture medium. The cells cultured by the invention can be propagated and passaged, the Shh signal channel is in an activated expression state, the tumorigenicity is not influenced, the cells can be frozen and stored at the temperature of minus 80 ℃, and the cells can be attached to the wall for differentiation, so that a unique cell model is provided for researching the molecular mechanism of the occurrence and development of the medulloblastoma and the molecular mechanism for regulating and controlling the Shh signal channel, and an important tool is provided for high-throughput screening of chemotherapeutic drugs.
Description
Technical Field
The invention belongs to the technical field of tissue cell culture, and particularly relates to a culture solution and a culture method for in-vitro culture of brain tumor cells.
Background
Medulloblastoma (MB) is the most common malignant brain tumor in children, which occurs mainly in the cerebellum and rapidly spreads into the central nervous system, and it is generally considered that MB includes at least 4 subtypes, the Wnt subtype, the Shh subtype, the third subtype and the fourth subtype. Of the 4 subtypes, Shh subtype accounts for about 30% of human medulloblastomas, and is caused by abnormal activation of Shh signaling pathway.
Astrocytes are the most widely distributed class of glial cells in the mammalian brain, and support the development and function of neurons. The role of astrocytes in brain tumors has also been elucidated to some extent in recent years: astrocytes have been reported to secrete Shh ligand in the context of neurodevelopment, adult neurogenesis, and brain injury. In medulloblastomas, Astrocytes promote the proliferation of tumor cells by secreting Shh protein (Liu, yongjiang, et. "assay protein production through hedgehog section." Cancer research 77.23(2017): 6692-6703.).
Due to the ease of culture and preservation, cell lines are widely used in cancer research. However, studies have shown that there is still a lack of an effective Shh-MB in vitro cell model. The Shh signaling pathway in cell lines currently used in either basic or preclinical studies of medulloblastoma is inactive. After knocking out the Shh pathway inhibitory protein Patched (Ptch1) by genetic means in the granular neuronal precursor cells in the mouse cerebellum, medulloblastoma was formed in the mouse brain. This model is currently widely used in basic as well as preclinical studies of medulloblastoma. However, studies have shown that the above tumor cells cultured by traditional adherent culture means are gradually inactivated in the Shh pathway in vitro, so that the tumor cells gradually stop proliferation, which brings great difficulty to the basic research of medulloblastoma and corresponding drug screening. A widely used adherent culture employs DMEM/10% FCS (Sasai, Ken, et al, "Shh pathway activity is down-regulated cultured medulloblastic cells: microorganisms for represented students," cancer research 66.8(2006): 4215-4222.).
Disclosure of Invention
The purpose of the present invention is to provide a culture solution and a culture method for in vitro cultured brain tumor cells, which are easy to culture in vitro, can be expanded and passaged, and in which the Shh signal pathway of cultured medulloblastoma cells is in an activated expression state and tumorigenicity is not affected.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention aims to provide a culture solution for culturing brain tumor cells in vitro, which comprises a nerve cell basic culture medium, B27 trophic factor with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, double-anti-penicillin/streptomycin with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, glutamine with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, sodium pyruvate with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, fetal bovine serum with the added mass of 0.5-1.5% of the mass of the nerve cell basic culture medium, and recombinant Shh (sonic hedgehog) protein. Wherein the addition amount of the recombinant Shh protein is 2.5 ug-3.5 ug added to each 1mL of the nerve cell basic culture medium.
Preferably, the addition amount of the recombinant Shh protein is 2.7 ug-3.3 ug per 1mL of the nerve cell basal medium.
Further preferably, the addition amount of the recombinant Shh protein is 2.8 ug-3.2 ug per 1mL of the neural cell basal medium.
Preferably, the addition mass of the fetal calf serum is 0.8-1.2% of the mass of the nerve cell basic culture medium.
Further preferably, the added mass of the fetal calf serum is 0.9-1.1% of the mass of the nerve cell basic culture medium.
Preferably, the addition mass of the B27 trophic factor is 1-3% of the mass of the nerve cell basic culture medium, the addition mass of the double-anti-penicillin/streptomycin is 0.5-1.5% of the mass of the nerve cell basic culture medium, the addition mass of the glutamine is 0.5-1.5% of the mass of the nerve cell basic culture medium, and the addition mass of the sodium pyruvate is 0.5-1.5% of the mass of the nerve cell basic culture medium.
More preferably, the addition mass of the B27 trophic factor is 1.5-2.5% of the mass of the nerve cell basic culture medium, the addition mass of the double-anti-penicillin/streptomycin is 0.9-1.1% of the mass of the nerve cell basic culture medium, the addition mass of the glutamine is 0.9-1.1% of the mass of the nerve cell basic culture medium, and the addition mass of the sodium pyruvate is 0.9-1.1% of the mass of the nerve cell basic culture medium.
The invention also aims to provide the application of the culture solution in the in vitro culture of the brain tumor cells.
Preferably, the brain tumor cell is a medulloblastoma cell.
Preferably, the in vitro culture method is suspension culture.
Preferably, the cell seeding density of the brain tumor cells in the culture solution is 3 × 105～5×105one/mL.
The third purpose of the invention is to provide an in vitro culture method of brain tumor cells, which inoculates the treated brain tumor cells into the culture solution for suspension culture.
Preferably, the cell seeding density is controlled to be 3 × 105～5×105one/mL.
Preferably, the brain tumor cell is a medulloblastoma cell.
Preferably, the medulloblastoma tissue extracted from the mouse cerebellum is digested for 20-40 min by using a digestive juice at 36-38 ℃, then the digestion is stopped by a pancreatin inhibitor, and then the treated brain tumor cell is obtained after filtration by a filter membrane and centrifugation, wherein each 10mL of the digestive juice contains the following components: 90U-110U of papain, 1 mg-3 mg of cysteine and 2400U-2600U of DNase.
In the present invention, the culture conditions for performing suspension culture are conventional conditions for cell culture.
The fourth purpose of the invention is to provide a tumor cell ball obtained by the in vitro culture method.
The fifth purpose of the invention is to provide a passage tumor cell, which is obtained by digesting the tumor cell sphere with digestive enzyme.
The sixth purpose of the invention is to provide the tumor cell ball cryopreservation method, wherein the tumor cell ball is cryopreserved by adopting a cryopreservation solution, the cryopreservation solution comprises serum and dimethyl sulfoxide in a mass ratio of 8-10: 1, and the cryopreservation temperature is-80 ℃.
In the present invention, cells obtained by culturing tumor cells extracted from mice in the culture medium of the present invention are referred to as primary cells; cells obtained by digesting cells cultured in a culture medium with a digestive juice and cells obtained by continuously culturing the cells are called passage cells.
The brain tumor cells cultured by the culture solution and the culture method converge into a sphere, which is called as a tumor cell sphere, so that the tumor cell sphere can survive for a long time and can be proliferated and passaged.
The invention adopts in vitro suspension culture, does not need to use polylysine (Poly-D-Lysine, PDL for short) for coating, and has simple and easy culture method.
When the culture solution and the culture method are adopted for in vitro culture, an Shh signal channel in the cell keeps an activated state, and the preclinical research of brain tumor is more convenient. Secondly, the tumor cells can be proliferated in a large amount in the culture solution and the culture method, and a large amount of cells are easily obtained for basic research and drug screening of brain tumors.
The primary cells and the passage cells cultured by the culture solution and the culture method of the invention keep tumorigenicity, and the separated tumor cell single cells or the cells digested by the tumor cell balls can grow medulloblastoma after being injected into the cerebellum of a mouse again. The method also enables us to preserve primary MB cells and passaged MB cells without changing tumorigenicity of tumor cells.
In addition, the tumor cell ball of the invention can be passaged, can be frozen and can be differentiated by attaching to the wall, and polylysine is adopted for coating during adherent differentiation.
The invention provides an important tool for researching the molecular mechanism of the occurrence and development of the medulloblastoma and screening the chemotherapeutic drugs aiming at the medulloblastoma in high flux. Meanwhile, a unique cell model is provided for researching the Shh signal channel regulation molecular mechanism.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the cells cultured by the culture solution and the culture method can be propagated and passaged, the Shh signal channel is in an activated expression state, the tumorigenicity is not influenced, the cells can be frozen at minus 80 ℃ and can be attached to the wall for differentiation, so that an important tool is provided for researching the molecular mechanism of the occurrence and development of the medulloblastoma and screening chemotherapeutic drugs aiming at the medulloblastoma at high flux. Meanwhile, a unique cell model is provided for researching the Shh signal channel regulation molecular mechanism.
The culture method is simple and easy to implement, and polylysine coating is not needed.
Drawings
FIG. 1 is a graph showing a comparison of the number of tumor cells per ml of culture solution after 3 days of culture with the addition of Shh protein;
FIG. 2 is a graph comparing the formation of tumor cells after 3 days of culture in different media;
FIG. 3 is a graph comparing the number of tumor cells per ml of culture medium cultured for 4days in different culture media;
FIG. 4 is a graph showing a comparison of cell viability values measured for MTT in different culture media for 2days, 3 days and 4 days;
FIG. 5 is a graph of immunocytochemical Ki67/Zic1 protein fluorescence against tumor spheres cultured for 4days in vitro;
FIG. 6 is a Tuj1 protein fluorescence plot of immunocytochemistry against tumor spheres cultured in vitro for 4 days;
FIG. 7 is a QPCR gene expression detection result chart of cultured tumor cells;
FIG. 8 is a diagram of a brain tumor of an immunodeficient mouse injected into a tumor;
FIG. 9 shows tumor cell spheres formed after passaging;
FIG. 10 shows tumor cell pellets after recovery.
Detailed Description
In order to make the present invention clearer, the present invention is further described with reference to the drawings and the embodiments, and it should be understood that the present embodiment is not intended to limit the scope of the present invention. Methods and conditions not described in detail in the present invention are conventional in the art.
Example 1
Experimental animals: conditionally knockout transgenic Mice Ptch1fl/fl Mice, Math1-Cre Mice and CB 17/SCIDMce, purchase in Jackson laboratories, all experimental animals meet SPF (Specific Pathogen Free), and all animal treatment operations are legally and effectively approved; immunodeficient Mice (CB17/SCID Mice) were implanted by intracranial injection using a mouse brain stereotaxic apparatus (KOPF, Nikon SMZ1000) and a 5ul microsampler (Shanghai Gao Pigeon).
Cell culture:
(1) and primary medulloblastoma cells are extracted from the mice brains of the conditioned knockout adult mice, the mouse cerebellar medulloblastoma tissues are digested by digestive juice, and the digestive juice components (10 ml): 100U of Papain (Papain, Worthington Biochemical, LS003126), 2mg of cysteine (L-cysteine, Sigma) and 2500U of DNase (DNase, Sigma, D4527) were digested in a water bath at 37 ℃ for 30 minutes. After termination of digestion with pancreatin inhibitor (Sigma, Roche,109878), single cell suspensions were obtained and passed through a 70um filter (Fisherbrand) and centrifuged to obtain treated medulloblastoma cells.
(2) And (2) inoculating the treated medulloblastoma cells obtained in the step (1) at a cell inoculation density of 4 × 105The inoculum size of each/mL is added into the culture solution for resuspension, and the suspension culture can be carried out in a 10cm culture dish or a six-hole plate, the culture condition is the conventional condition of cell culture (37 ℃/CO2/O2 constant temperature incubator), the culture solution does not need to be replaced or supplemented within 4days of the formation of the tumor cells, the volume of the tumor cells is not too large, the culture can be carried out for 5 days generally, and the digestion and subculture can be carried out continuously. Culture broth (i.e., NB-Shh-B27-1% FBS): the nerve cell basal medium (Neurobasal medium is abbreviated as NB, Invitrogen), 3ug of recombinant Shh protein (ab75431, abcam), B27 trophic factor (Invitrogen) accounting for 2% of the mass of the nerve cell basal medium, double anti-penicillin/streptomycin (Pen/Strep, Invitrogen) accounting for 1% of the mass of the nerve cell basal medium, glutamine (L-glutamine, Invitrogen) accounting for 1% of the mass of the nerve cell basal medium, sodium pyruvate (Na-Pyrrate, Invitrogen) accounting for 1% of the mass of the nerve cell basal medium, and Fetal Bovine serum (Fetal Bovine serum, Able, Atlanta Biologicals, S11150) accounting for 1% of the mass of the nerve cell basal medium are added to 1mL of the nerve cell basal medium.
Comparative example 1
The procedure was substantially the same as in example 1, except that no recombinant Shh protein (i.e., NB-B27-1% FBS) was added to the culture broth in example 1.
Comparative example 2
The procedure was substantially the same as in example 1, except that the fetal bovine serum was added to the culture medium in an amount of 3% by mass based on the amount of the nerve cell basal medium in example 1 (i.e., NB-Shh-B27-3% FBS).
Comparative example 3
Substantially the same as example 1, except that: the culture medium was neural Stem Cell medium (Mouse, Stem Cell Technologies) containing Proliferation Supplement 10% of the neural Stem Cell medium (Proliferation Supplement, Mouse, Stem Cell Technologies,05701), EGF (Epidermal Growth Factor, Stem Cell Technologies,78006) at 20ng/ml, FGF (Fibroblast Growth Factor, Stem Cell Technologies,78003) (i.e., NSC media).
Comparative example 4
Substantially the same as example 1, except that: the medium was DMEM (life technologies, Gibco,11995073) which is a common basal medium containing 10% fetal bovine serum (i.e., DMEM-10% FBS) by mass of the common basal medium.
Detection and data
1. Comparing the formation and cell viability of the tumor cells under the culture conditions of example 1 and comparative examples 1 to 4, wherein the formation number of the tumor cells after 3 days of culture by using the culture solution of comparative example 1 and example 1 is shown in the sequence from left to right in FIG. 1; FIG. 2 shows the numbers of spheroids and the sizes of the spheroids of the tumor cells cultured in different culture media for 3 days, wherein the upper left is the case of culturing the culture medium of example 1, the upper right is the case of culturing the culture medium of comparative example 2, the lower left is the case of culturing the culture medium of comparative example 3, and the lower right is the case of culturing the culture medium of comparative example 4; FIG. 3 is a graph showing the number of tumor cells per ml of a culture solution cultured for 4days in different culture solutions, in which four columns represent, from left to right, the culture conditions of the culture solution of example 1, the culture solution of comparative example 2, the culture solution of comparative example 3, and the culture solution of comparative example 4, respectively; FIG. 4 shows the viability values of cells cultured in different culture media for 2days, 3 days and 4days, respectively, in MTT assay, in which NB-1% FBS represents the examples1, NB-3% FBS represents the culture of comparative example 2, NSC media represents the culture of comparative example 3, and the cell viability rate of example 1 after 4days of culture is significantly higher than that of example 3. Wherein, the cell viability is measured by adopting an MTT method (MTT, trade name thiazole blue, chemical name 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt), and the measuring method is as follows: after the spherocytes were digested, centrifuged, counted and the cell density of each experimental group was ensured to be the same, 10ul MTT (5mg/ml) was added to each well, 3 replicates per group, 37 ℃/CO2/O2After incubation in a constant temperature incubator for 4 hours, MTT was removed by centrifugation, 100ul of dimethyl sulfoxide was added, the resulting mixture was placed in the incubator for 2 hours to sufficiently dissolve crystals, and absorbance was measured at 595nm using an enzyme reader (model No. imark 15580).
As can be seen from fig. 1 to 4, the primary MB tumor cell cultured in example 1 was most effective, while the culture in comparative examples 1 to 4 was not effective, as compared to the analysis of the number of formed tumor cells and cell viability.
2. Cultured for 4days according to the method of example 1, immunocytochemistry and cytobiology analysis: a first antibody: ki67(1:500, Abcam), Tuj 1(1:500, Abcam), Zic1(1:500, Abcam); secondary antibody: goat anti-Mouse Alexa Fluor-594anti-Mouse IgG (1:200), goat anti-Rabbit Alexa Fluor-594anti-Rabbit IgG (1:200), stained using conventional SP immunocytochemistry, and all immunofluorescent cell samples were photographed using a fluorescence inverted Microscope (Nikon Eclipse Ti Microscope), and the results are shown in FIGS. 5 and 6. From fig. 5 and fig. 6, it can be seen that the cells in the tumor sphere express the cell proliferation marker Ki67 protein, the marker Zic1 protein expressing cerebellar cells and the marker Tuj1 protein expressing neurons. Thus, the tumor cell balls in this culture condition still have their original neural properties and proliferation properties.
3. Culturing for 2 to 4days according to the method of example 1, and culturing for 1 to 2days according to a traditional adherent culture method, wherein the traditional adherent culture method comprises the following steps: PDL was first coated onto the well plates, then NB-Shh-B27/1% FBS medium was resuspended and placed at 37 ℃/CO2/O2Culturing in a constant temperature incubator. In order to eliminate the possible influence of the Shh protein and the serum on the Shh signal channel in the experiment, the experiment is carried outThe culture solution under homospheronization culture, namely NB-Shh-B27/1% FBS, is adopted as the nutrient solution, Shh protein and FBS are added, and then the activation condition of the GLi1/Ptch2/Sfrp1 gene in the Shh signal channel is detected by a real-time fluorescence quantification-PCR (QPCR) method.
Real-time fluorescent quantitative PCR: RNA in cell samples was extracted by lysis with Trizol reagent, followed by reverse transcription to synthesize cDNA according to standard procedures provided in the reverse transcription kit Qiagen RT kit, and quantitative PCR detection analysis (BIORAD iCycler iQ system).
Generally, the Shh signal pathway of a just-extracted primary tumor cell is in an activated state, compared with an adherent differentiated tumor cell and a just-extracted primary tumor cell, the expression of Shh signal pathway target genes is obviously reduced after the traditional adherent culture of the primary tumor cell is cultured for 2days, the Shh signal pathway is gradually inactivated, and the expression of the three genes is still maintained at a higher level when the tumor cell is cultured in vitro for 4days, which indicates that the Shh signal pathway is in an activated state, and the result is shown in fig. 7, wherein 0hr is the gene activation condition of the just-extracted primary tumor cell; 1day adhesive culture is the gene activation condition of traditional adherent culture for 1 day; 2days for traditional adherent culture, wherein the gene activation condition is 2days for traditional adherent culture; 2days cultivation for 2days gene activation as in example 1; the 4days sphere culture refers to the gene activation in the 4days culture in example 1.
4. After 4days of culture according to the method of example 1, they were divided into two groups, the first group was injected directly into the cerebellum of CB17-SCID mice without treatment, and the second group was injected into the cerebellum of CB17-SCID mice after digesting into single cells using the digestive enzyme Accutase. It can be seen that the injection of two groups of cells into the mouse cerebellum all produce medulloblastoma, which indicates that the medulloblastoma cultured by the method has tumorigenicity, and the culture mode of the tumor cell can be used for preclinical research of therapeutic drugs such as proliferation inhibition and the like. The results are shown in fig. 8, the brain tumor map of the injection tumor (left one is intracranial tumor brain map of the injection two-week immunodeficient mice, and hematoxylin-eosin (HE) stained section map of the right two-week tumor cerebellum) and table 1.
TABLE 1 statistics of tumor formation
| Class of injected cells | Number of long tumor mice (one)/total amount of mice (one) | Tumor formation rate (%) |
| Tumor |
3/3 | 100 |
| Digested |
3/3 | 100 |
5. Using a digestive enzyme: accutase can be used for subculturing after digesting the tumor sphere cells into single cells, and FIG. 9 shows the generation 2 tumor sphere cells formed after digesting and subculturing the 4-day cultured tumor sphere cells.
6. Using a freezing medium: 90% serum/10% dimethyl sulfoxide, the tumor cell can be frozen at-80 ℃ for 4days, fig. 10 shows that the tumor cell can be successfully recovered after being frozen at-80 ℃ for 4days, and the tumor cell growing for the next day shows that the tumor cell has the capability of low-temperature preservation, while the traditional primary culture medulloblastoma cell can not be frozen at low temperature, which is another great advantage of primary spheroidization culture.
Claims (8)
1. A culture solution for culturing brain tumor cells in vitro is characterized in that: the culture solution comprises a nerve cell basic culture medium, B27 trophic factors with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, bis-anti-penicillin/streptomycin with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, glutamine with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, sodium pyruvate with the added mass of 0.5-5% of the mass of the nerve cell basic culture medium, fetal calf serum with the added mass of 0.5-1.5% of the mass of the nerve cell basic culture medium, and recombinant Shh protein; wherein the addition amount of the recombinant Shh protein is 2.5-3.5 mu g per 1mL of the nerve cell basic culture medium.
2. The culture solution for in vitro culturing of brain tumor cells according to claim 1, wherein: the addition amount of the recombinant Shh protein is 2.8-3.2 mu g of the recombinant Shh protein added into every 1mL of the nerve cell basic culture medium.
3. The culture solution for in vitro culturing of brain tumor cells according to claim 1, wherein: the addition mass of the fetal calf serum is 0.8-1.2% of the mass of the nerve cell basic culture medium.
4. Use of a culture solution according to any one of claims 1 to 3 for the in vitro culture of brain tumor cells; the brain tumor cell is a medulloblastoma cell.
5. Use according to claim 4, characterized in that: the in vitro culture method is suspension culture.
6. The use of claim 4, wherein said brain tumor cells are seeded in said culture medium at a cell seeding density of 3 × 105~5×105one/mL.
7. An in vitro culture method of brain tumor cells, which is characterized in that: inoculating the treated brain tumor cells into the culture solution of any one of claims 1 to 3 for suspension culture; the brain tumor cell is a medulloblastoma cell.
8. The method for culturing brain tumor cells according to claim 7, wherein: the preparation method of the treated brain tumor cell comprises the following steps: digesting a medulloblastoma tissue extracted from a mouse cerebellum for 20-40 min at 36-38 ℃ by using a digestive juice, stopping digestion by using a pancreatin inhibitor, filtering by using a filter membrane, and centrifuging to obtain the treated brain tumor cells, wherein each 10mL of the digestive juice contains the following components: 90-110U of papain, 1-3 mg of cysteine and 2400-2600U of DNase.
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