CN110295145A - A kind of culture solution and cultural method of in vitro culture brain tumor cell - Google Patents
A kind of culture solution and cultural method of in vitro culture brain tumor cell Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
Abstract
The present invention provides a kind of culture solutions of in vitro culture brain tumor cell, culture solution includes nerve cell basal medium, is that 0.5%~5% B27 trophic factors, 0.5%~5% dual anti-- penicillin/streptomycin, 0.5%~5% glutamine, 0.5%~5% Sodium Pyruvate, 0.5%~1.5% fetal calf serum, the 2.5ug/mL~3.5ug/mL of nerve cell basal medium quality recombinate Shh albumen.The cell that culture of the present invention obtains can be proliferated passage, Shh signal path is in activation expression status, oncogenicity is not affected by influence, -80 DEG C are able to bear to freeze, adherent it can break up, to for research medulloblastoma occurrence and development molecular mechanism and Shh signal path regulatory molecule mechanism provides unique cell model and high flux screening chemotherapeutics provides important tool.
Description
Technical field
The invention belongs to histocyte culture technique fields, and in particular to a kind of culture solution of in vitro culture brain tumor cell
And cultural method.
Background technique
Medulloblastoma (Medulloblastoma, abbreviation MB) is the most common malignant brain tumor in children, it mainly goes out
It in present cerebellum, and can rapidly diffuse into central nervous system, it is generally accepted that MB includes at least 4 kinds of hypotypes, respectively Wnt
Hypotype, Shh hypotype, third hypotype and the 4th hypotype.In 4 kinds of hypotypes, Shh hypotype accounts for about the 30% of people's medulloblastoma, by Shh
The abnormal activation of signal path and cause.
Astroglia is the widest a kind of spongiocyte of mammal intracerebral distribution, development and function to neuron
Supporting function can be played.Effect of the astroglia in brain tumor in recent years also obtains elaboration to a certain extent: having been reported that
It was found that astroglia secretes Shh ligand in the case where neurodevelopment, adult neural's forming region and cerebral injury.In marrow
In blastoma, astroglia promotes proliferation (Liu, Yongqiang, the et of tumour cell by secretion Shh albumen
al."Astrocytes promote medulloblastoma progression through hedgehog
secretion."Cancer research 77.23(2017):6692-6703.)。
Due to being easy to cultivate preservation, cell strain is widely used in the research of cancer.But it studies have shown that still lacks at present
Weary effective Shh-MB In vitro cell model.Currently used in the cell strain of medulloblastoma basic research or preclinical study
Shh signal path is all in inactivated state.It is logical to knock out Shh in Granule Neurons precursor in mouse cerebellum with science of heredity means
After the repressible protein Patched (Ptch1) on road, medulloblastoma is formed in the brain of mouse.The model is widely used at present
In the basic research of medulloblastoma and preclinical study.However, studies have shown that being cultivated with traditional adhere-wall culture means
Above-mentioned tumour cell Shh access gradually inactivates in vitro so that tumour cell tapers off proliferation, this is to medulloblastoma
Basic research and relative medicine screening bring very big difficulty.Culture medium used by the adhere-wall culture being widely used is
DMEM/10%FCS (Sasai, Ken, et al. " Shh pathway activity is down-regulated in
cultured medulloblastoma cells:implications for preclinical studies."Cancer
research 66.8(2006):4215-4222.)。
Summary of the invention
It is easy in vitro culture the purpose of the present invention is to provide one kind and passage can be proliferated, the medulloblastoma after culture
The Shh signal path of cell is in activation expression status and oncogenicity is not affected by the culture of the in vitro culture brain tumor cell of influence
Liquid and cultural method.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
It is an object of the present invention to provide a kind of culture solution of in vitro culture brain tumor cell, the culture solution includes
Nerve cell basal medium, 0.5%~5% B27 that addition quality is the nerve cell basal medium quality are sought
Support the factor, 0.5%~5% dual anti-- penicillin/strepto- that addition quality is the nerve cell basal medium quality
Element, addition quality are 0.5%~5% glutamine of the nerve cell basal medium quality, addition quality is institute
0.5%~5% Sodium Pyruvate of the nerve cell basal medium quality stated, addition quality are the nerve cell basis
0.5%~1.5% fetal calf serum, recombination Shh (Sonic hedgehog) albumen of culture medium quality.Wherein, the weight
The additive amount of group Shh albumen is that 2.5ug~3.5ug is added in nerve cell basal medium described in every 1mL.
Preferably, the additive amount of the recombination Shh albumen is that nerve cell basal medium described in every 1mL is added
2.7ug~3.3ug.
It is further preferred that the additive amount of the recombination Shh albumen is nerve cell basal medium described in every 1mL
2.8ug~3.2ug is added.
Preferably, the addition quality of the fetal calf serum is the 0.8% of the nerve cell basal medium quality
~1.2%.
It is further preferred that the addition quality of the fetal calf serum is the nerve cell basal medium quality
0.9%~1.1%.
Preferably, the addition quality of the B27 trophic factors is the 1% of the nerve cell basal medium quality
~3%, the addition quality of the dual anti-- penicillin/streptomycin is the 0.5% of the nerve cell basal medium quality
~1.5%, the addition quality of the glutamine is the 0.5%~1.5% of the nerve cell basal medium quality,
The addition quality of the Sodium Pyruvate is the 0.5%~1.5% of the nerve cell basal medium quality.
It is further preferred that the addition quality of the B27 trophic factors is the nerve cell basal medium matter
The 1.5~2.5% of amount, the addition quality of the dual anti-- penicillin/streptomycin are the nerve cell basal medium matter
The 0.9%~1.1% of amount, the addition quality of the glutamine are the nerve cell basal medium quality
0.9%~1.1%, the addition quality of the Sodium Pyruvate be the nerve cell basal medium quality 0.9%~
1.1%.
It is a further object to provide the answering in the in vitro culture of brain tumor cell of the culture solution described in one kind
With.
Preferably, the brain tumor cell is medulloblastoma cell.
Preferably, the method for the in vitro culture is the culture that suspends.
Preferably, cell-seeding-density of the brain tumor cell in the culture solution is 3 × 105~5 × 105
A/mL.
It, will treated brain tumor third object of the present invention is to provide a kind of extracorporeal culturing method of brain tumor cell
Cell inoculation carries out suspension culture into above-mentioned culture solution.
Preferably, control cell-seeding-density is 3 × 105~5 × 105A/mL.
Preferably, the brain tumor cell is medulloblastoma cell.
Preferably, it will be digested at 36 DEG C~38 DEG C using digestive juice from the pith mother cells tumor tissue extracted in mouse cerebellum
Then 20~40min is terminated with pancreatin inhibitor and is digested, treated the brain is obtained then through membrane filtration, after centrifugation
Tumour cell, wherein the ingredient in digestive juice described in every 10mL is as follows: the papain of 90U~110U, 1mg~3mg
The DNA enzymatic of cysteine and 2400U~2600U.
In the present invention, the condition of culture for carrying out suspension culture is the normal condition of cell culture.
Fourth object of the present invention is to provide a kind of tumour cell obtained using the extracorporeal culturing method culture
Ball.
Fifth object of the present invention is to provide a kind of passage of tumor glomus cells, by the tumour cell ball using digestion
Enzymic digestion obtains.
Sixth object of the present invention is to provide the cryopreservation methods of the tumour cell ball described in one kind, using frozen stock solution to institute
The tumour cell ball stated is frozen, and the frozen stock solution includes the serum and dimethyl sulfoxide that mass ratio is 8~10:1, can be frozen
Temperature is -80 DEG C.
In the present invention, we will be equal from the cell of the culture solution culture in the tumour cell present invention extracted in mouse
Referred to as primary cell；After cell that cell after culture solution culture is digested through digestive juice and the cell are continued culture
Cell is known as passage cell.
Using culture solution of the invention and the brain tumor cell of cultural method culture meeting glomeration shape, we term it swollen
Oncocyte ball enables the tumour cell ball to survive and be proliferated for a long time passage.
The present invention uses In vitro Suspension culture, without using poly-D-lysine (Poly-D-Lysine, abbreviation PDL) packet
Quilt, cultural method are simple and easy.
When carrying out in vitro culture using culture solution of the invention and cultural method, the Shh signal path in cell keeps living
Change state, more convenient for brain tumor preclinical study.Secondly, tumour cell can be a large amount of in above-mentioned culture solution and cultural method
Proliferation is easily obtained basic research and drug screening that a large amount of cell carries out brain tumor.
Tumor formation is kept using the primary cell and passage cell of culture solution of the invention and cultural method culture, will be divided
From tumour ball individual cells or the postdigestive cell of tumour cell ball be re-injected into after mouse cerebellum and can grow marrow
Blastoma.The method, also can let us in the case where not changing tumour cell tumorigenicity, save primary MB cell and
The MB cell of passage.
In addition, tumour cell ball of the invention can be passed on, can be frozen and adherent can break up, when adherent differentiation, is relied using poly
Propylhomoserin coating.
The present invention is the molecular mechanism for studying medulloblastoma occurrence and development and high flux screening for medulloblastoma
Chemotherapeutics provide important tool.Meanwhile it also providing for research Shh signal path regulatory molecule mechanism unique thin
Born of the same parents' model.
Due to the implementation of above technical scheme, the invention has the following advantages over the prior art:
The cell that culture solution and cultural method culture of the invention obtains can be proliferated passage, and Shh signal path, which is in, to swash
Expression status living, oncogenicity are not affected by influence, are able to bear -80 DEG C and freeze, adherent can break up, thus for research medulloblastoma
The molecular mechanism and high flux screening of occurrence and development provide important tool for the chemotherapeutics of medulloblastoma.Together
When, also unique cell model is provided for research Shh signal path regulatory molecule mechanism.
Cultural method of the invention is simple and easy, is coated with without using poly-D-lysine.
Detailed description of the invention
Fig. 1 is the Tumor sphere cell quantitative comparison figure after being added the culture of Shh albumen 3 days in every milliliter of culture solution；
Fig. 2 is the formational situation comparison diagram of Tumor sphere cell after cultivating 3 days under different culture solutions；
Fig. 3 is the Tumor sphere cell quantitative comparison figure in every milliliter of culture solution for cultivate under different culture solutions 4 days；
Fig. 4 is that MTT is measured and cultivated 2 days, 3 days, 4 days cell viability value comparison diagrams in different culture solutions respectively；
Fig. 5 is the immunocytochemistry Ki67/Zic1 protein fluorescence figure that is carried out in vitro culture 4 days tumour balls；
Fig. 6 is is carried out 1 protein fluorescence figure of immunocytochemistry Tuj in vitro culture 4 days tumour balls；
Fig. 7 is the tumour cell QPCR gene expression detection result figure of culture；
Fig. 8 is the immunodeficient mouse brain tumor figure for injecting tumor formation；
Fig. 9 is the tumour cell ball formed after passing on；
Figure 10 is the tumour cell ball after recovery.
Specific embodiment
In order to keep the present invention clearer, the present invention will be further described in conjunction with the accompanying drawings and embodiments, it should be understood that this
Embodiment is not intended to limit the scope of protection of the present invention.The method and condition being not described in detail in the present invention is the normal of this field
Rule condition.
Embodiment 1
Experimental animal: conditionity knocks out trangenic mice Ptch1fl/fl Mice, Math1-Cre Mice, CB17/SCID
Mice, is purchased from the laboratory Jackson, and all animal for research meet SPF grades of (Specific Pathogen Free, no spies
Determine pathogen), all animal processing operations pass through legal effective approval；Immunodeficient mouse (CB17/SCID Mice), cranium
Interior injection transplantation uses mouse stereotaxic apparatus (KOPF, Nikon SMZ1000) and 5ul microsyringe (Shanghai Gao Ge).
Cell culture:
(1), primary medulloblastoma cell is knocked out in adult mice cerebellum from above-mentioned condition and is extracted, mouse cerebellum marrow
Blastoma tissue using digestive juice digest, digestive juice ingredient (10ml): 100U papain (Papain,
Worthington Biochemical, LS003126), the cysteine (L-cysteine, Sigma) of 2mg and 2500U
DNA enzymatic (DNase, Sigma, D4527), 37 DEG C of water-baths digest 30 minutes.Pancreatin inhibitor (Sigma, Roche, 109878) is eventually
It after only digesting, obtains single cell suspension and crosses 70um filter membrane (Fisherbrand), obtain that treated that medulloblastoma is thin after centrifugation
Born of the same parents.
(2), by step (1) medulloblastoma cell that obtains that treated by cell-seeding-density 4 × 105A/mL's
Inoculum concentration is added in culture solution and is resuspended, and can carry out suspension culture in 10cm culture dish or six orifice plates, condition of culture is cell culture
Normal condition (37 DEG C/CO2/O2 constant incubator), Tumor sphere cell formed 4 days in do not need replace or supplement training
Nutrient solution, Tumor sphere cell volume should not be too large, and general culture can carry out had digestive transfer culture and continue to cultivate to 5 days.Culture solution (i.e. NB-
Shh-B27-1%FBS): nerve cell basal medium (Neurobasal medium abbreviation NB, Invitrogen), every 1mL
Nerve cell basal medium is added 3ug recombination Shh albumen (ab75431, abcam), accounts for nerve cell basal medium quality
2% B27 trophic factors (Invitrogen), account for 1% dual anti-- penicillin/chain of nerve cell basal medium quality
Mycin (Pen/Strep, Invitrogen), the 1% glutamine (L- for accounting for nerve cell basal medium quality
Glutamine, Invitrogen), account for nerve cell basal medium quality 1% Sodium Pyruvate (Na-Pyrurate,
Invitrogen), 1% fetal calf serum (the Fetal Bovine Serum of nerve cell basal medium quality is accounted for
Premium, abbreviation FBS, Atlanta Biologicals, S11150).
Comparative example 1
Substantially same as Example 1, difference, which is only that in embodiment 1 in culture solution, does not add recombination Shh albumen (culture solution
That is NB-B27-1%FBS).
Comparative example 2
Substantially same as Example 1, difference is only that the additive amount of the fetal calf serum in embodiment 1 in culture solution accounts for nerve
3% (i.e. NB-Shh-B27-3%FBS) of Cell Basal Medium quality.
Comparative example 3
Substantially same as Example 1, difference is: culture solution is nerve stem cell culture medium (NeuroCult Basal
Medium, Mouse, Stem Cell Technologies), add containing 10% proliferation for accounting for nerve stem cell culture medium quality
Add object (Proliferation Supplement, Mouse, Stem Cell Technologies, 05701), 20ng/ml's
EGF (Epidermal Growth Factor, Stem Cell Technologies, 78006), the FGF of 10ng/ml
(Fibroblast Growth Factor, Stem Cell Technologies, 78003) (i.e. NSC media).
Comparative example 4
Substantially same as Example 1, difference is: culture solution is conventional foundation culture solution DMEM (Life
Technologies, Gibco, 11995073), contain 10% fetal calf serum (the i.e. DMEM- for accounting for conventional foundation culture medium quality
10%FBS).
Detection and data
1, the formational situation of the Tumor sphere cell under the condition of culture of comparing embodiment 1 and comparative example 1 to 4 and cell are living
Power, wherein Fig. 1 is followed successively by from left to right forms number using Tumor sphere cell behind comparative example 1, culture solution culture 3 days of embodiment 1
Measure situation；Fig. 2 is the balling-up quantity and tumour ball size of Tumor sphere cell after cultivating 3 days under different culture solutions, wherein the picture left above
For the culture situation of the culture solution of embodiment 1, top right plot is the culture situation of the culture solution of comparative example 2, and lower-left figure is comparative example 3
Culture solution culture situation, bottom-right graph be comparative example 4 culture solution culture situation；Fig. 3 is to cultivate 4 days under different culture solutions
Every milliliter of culture solution in Tumor sphere cell quantity situation, wherein four pillars respectively represent the training of embodiment 1 from left to right
The culture situation of nutrient solution, the culture situation of the culture solution of comparative example 2, the culture situation of the culture solution of comparative example 3, comparative example 4
The culture situation of culture solution；Fig. 4 is that MTT is measured and cultivated 2 days, 3 days, 4 days cell viability value situations in different culture solutions respectively,
Wherein, what NB-1%FBS was indicated is the culture situation of embodiment 1, and what NB-3%FBS was indicated is the culture situation of comparative example 2,
What NSC media was indicated is the culture situation of comparative example 3, and the Cell viability that embodiment 1 is cultivated 4 days is apparently higher than the thin of ratio 3
Born of the same parents' motility rate value.Wherein, cell viability measurement uses mtt assay (MTT, trade name thiazolyl blue, chemical name 3- (4,5- dimethylthiazoles-
2) -2,5- diphenyltetrazolium bromide bromide), measuring method is as follows: glomus cell is digested, is centrifuged, is counted and ensures each experimental group cell
After density is identical, every hole is added 10ul MTT (5mg/ml), every group of 3 multiple holes, and 37 DEG C/CO2/O2It is small that constant incubator is incubated for 4
Shi Hou, centrifugation removal MTT, is added 100ul dimethyl sulfoxide, is put into incubator 2 hours with abundant dissolving crystallized body, microplate reader
(model NO.IMark 15580) 595nm measures absorbance.
Compare from Fig. 1 to Fig. 4 as it can be seen that being analyzed from the forming quantity and cell viability of Tumor sphere cell, embodiment 1 is cultivated
Primary MB Tumor sphere cell effect it is best, and the culture effect of comparative example 1 to 4 is bad.
2, it cultivates 4 days according to the method for embodiment 1, immunocytochemistry and cytogenetic analysis: primary antibody: Ki67 (1:
500, Abcam), Tuj 1 (1:500, Abcam), Zic1 (1:500, Abcam)；Secondary antibody: sheep anti mouse Alexa Fluor-
594anti-Mouse IgG (1:200), goat-anti rabbit Alexa Fluor-594anti-Rabbit IgG (1:200), using routine
The dyeing of SP immunocytochemistry, the shooting of all immunofluorescence cell samples are all made of fluorescence inverted microscope shooting
(Nikon Eclipse Ti Microscope), is as a result shown in Fig. 5, Fig. 6.It can be seen that the cell table in tumour ball from Fig. 5, Fig. 6
Up to cell proliferation marker Ki67 albumen, the marker Zic1 albumen of cerebellar cell and the marker Tuj of expression neuron are expressed
1 albumen.Illustrate, the tumour cell ball under the condition of culture still has its original neural characteristic, proliferative.
3, it cultivates 2 to 4 days according to the method for embodiment 1, according to method culture 1 to 2 day of traditional adhere-wall culture, wherein
Traditional adhere-wall culture method are as follows: first PDL is coated with orifice plate, and rear NB-Shh-B27/1%FBS culture solution is resuspended, be placed in 37 DEG C/
CO2/O2Constant incubator culture.It influences, therefore cultivates to exclude Shh albumen and serum to the possibility of Shh signal path in the experiment
Liquid is used with the culture solution under balling-up culture, i.e. NB-Shh-B27/1%FBS, joined Shh albumen and FBS, then with real-time
The Activation of GLi1/Ptch2/Sfrp1 gene in the method detection Shh signal path of Fluorescent Quantitative-PCR (QPCR).
Real-time fluorescence quantitative PCR: the RNA extracted in cell sample is cracked by Trizol reagent, then according to reverse transcription
The standard operation that kit Qiagen RT kit is provided carries out reverse transcription and synthesizes cDNA, and carries out quantitative PCR detection analysis
(BIORAD iCycler iQ system)。
It is generally believed that the primary tumor cell Shh signal path just extracted is the state of activation, the tumour with adherent differentiation
Cell and the primary tumor cell just extracted are compared, and the primary tumor cell of traditional adhere-wall culture shh signal after culture 2 days is logical
The expression of road target gene is lowered obvious, and shh signal path gradually inactivates, and at Tumor sphere cell in vitro culture 4 days, this three bases
The expression of cause still maintains higher level, shows that Shh signal path is active, as a result sees Fig. 7, wherein 0hr is just to mention
The gene activation situation of the primary tumor cell taken；1days adherent culture is 1 day gene of traditional adhere-wall culture
Activation；2days adherent culture is 2 days gene activation situations of traditional adhere-wall culture；2days sphere
Culture is method culture 2 days gene activation situations of embodiment 1；4days sphere culture is the side of embodiment 1
Method culture 4 days gene activation situations.
4, it cultivates 4 days according to the method for embodiment 1, by two groups of its point, first group does not deal with and be injected directly into CB17-
SCID mice cerebellum, second group uses digestive ferment Accutase, and being digested to individual cells, to re-inject into CB17-SCID mouse small
Brain.It can be seen that two groups of cell infusions generate medulloblastoma to mouse cerebellum, illustrate the medulloblastoma tool of the method culture
There is oncogenicity, illustrates that the training method of the Tumor sphere cell can be used for the preclinical of the chemotherapeutics such as Inhibit proliferaton and grind again
Study carefully.As a result as the brain tumor figure of Fig. 8 injection tumor formation (the first from left is two weeks immunodeficient mouse encephalic tumor formation mind maps of injection, right two at
Hematoxylin-eosin staining procedures (HE) stained slice figure of tumor cerebellum) and table 1.
1 tumor formation situation statistical form of table
| Inject cell class | Bearing tumor mouse number (only)/mouse total amount (only) | Tumor formation rate (%) |
| Tumor sphere cell | 3/3 | 100 |
| Postdigestive single Tumor sphere cell | 3/3 | 100 |
5, using digestive ferment: it is culture that Accutase, which digests Tumor sphere cell at that can carry out secondary culture, Fig. 9 after unicellular,
The 2nd generation Tumor sphere cell that 4 days Tumor sphere cells are formed after had digestive transfer culture.
6, using frozen stock solution: 90% serum/10% dimethyl sulfoxide, Tumor sphere cell can freeze in -80 DEG C of refrigerators, Figure 10
Successful resuscitation is carried out after freezing 4 days for -80 DEG C, two days Tumor sphere cells of growth regulation illustrate that the Tumor sphere cell has low temperature
The ability of preservation, and traditional originally culture medulloblastoma cell is still impatient at cryopreservation, this is primary balling-up culture
Another big advantage.
Claims (12)
1. a kind of culture solution of in vitro culture brain tumor cell, it is characterised in that: the culture solution includes nerve cell basis
Culture medium, addition quality are 0.5%~5% B27 trophic factors of the nerve cell basal medium quality, addition matter
Measure 0.5%~5% dual anti-- penicillin/streptomycin for the nerve cell basal medium quality, addition quality is institute
0.5%~5% glutamine of the nerve cell basal medium quality stated, addition quality are the nerve cell basis
0.5%~5% Sodium Pyruvate of culture medium quality, addition quality are the nerve cell basal medium quality
0.5%~1.5% fetal calf serum recombinates Shh albumen.Wherein, the additive amount of the recombination Shh albumen is described in every 1mL
Nerve cell basal medium be added 2.5ug~3.5ug.
2. the culture solution of in vitro culture brain tumor cell according to claim 1, it is characterised in that: the recombination Shh
The additive amount of albumen is that 2.8ug~3.2ug is added in nerve cell basal medium described in every 1mL.
3. the culture solution of in vitro culture brain tumor cell according to claim 1, it is characterised in that: the fetal calf serum
Addition quality be the nerve cell basal medium quality 0.8%~1.2%.
4. a kind of application of culture solution as claimed any one in claims 1 to 3 in the in vitro culture of brain tumor cell.
5. application according to claim 4, it is characterised in that: the brain tumor cell is medulloblastoma cell.
6. application according to claim 4, it is characterised in that: the method for the in vitro culture is the culture that suspends.
7. application according to claim 4, it is characterised in that: the brain tumor cell is thin in the culture solution
Born of the same parents' inoculum density is 3 × 105~5 × 105A/mL.
8. a kind of extracorporeal culturing method of brain tumor cell, it is characterised in that: by treated, brain tumor cell is seeded to right
It is required that carrying out suspension culture in culture solution described in any one of 1 to 3.
9. the extracorporeal culturing method of brain tumor cell according to claim 8, it is characterised in that: treated the brain
Tumour cell the preparation method comprises the following steps: by from the pith mother cells tumor tissue extracted in mouse cerebellum at 36 DEG C~38 DEG C using digestion
Liquid digests 20~40min, is then terminated and is digested with pancreatin inhibitor, and then through membrane filtration, the processing is obtained after centrifugation
Brain tumor cell afterwards, wherein the ingredient in digestive juice described in every 10mL is as follows: papain, the 1mg of 90U~110U
The cysteine of~3mg and the DNA enzymatic of 2400U~2600U.
10. a kind of tumour cell ball obtained using extracorporeal culturing method culture described in any one of claim 8 to 9.
11. a kind of passage of tumor glomus cell, it is characterised in that: tumour cell ball described in any one of claim 10 is disappeared using digestive ferment
Change and obtains.
12. a kind of cryopreservation methods of tumour cell ball as claimed in claim 10, it is characterised in that: using frozen stock solution to described
Tumour cell ball frozen, the frozen stock solution include mass ratio be 8~10:1 serum and dimethyl sulfoxide.
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| WO2014011881A2 (en) * | 2012-07-11 | 2014-01-16 | Imstem Biotechnology, Inc. | Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof |
| WO2017160234A1 (en) * | 2016-03-14 | 2017-09-21 | Agency For Science, Technology And Research | Generation of midbrain-specific organoids from human pluripotent stem cells |
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| WO2014011881A2 (en) * | 2012-07-11 | 2014-01-16 | Imstem Biotechnology, Inc. | Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof |
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