CN111979197A - Glioma stem cell in-vitro culture method and culture medium - Google Patents
Glioma stem cell in-vitro culture method and culture medium Download PDFInfo
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Abstract
The invention provides a glioma stem cell in-vitro isolated culture method and a culture medium, wherein the method comprises the following steps: the first step is as follows: adding the cytoma tissue into PBS buffer solution; removing blood clots and necrotic cells, and cutting into paste; the second step is that: adding an Accutase solution to digest the tissue blocks into cell suspension; the third step: adding GC glioma cell culture medium, mixing uniformly, and stopping digestion; filtering the cell suspension by a filter screen (BD) to obtain a single cell suspension; the fourth step: adding PBS to re-suspend the precipitate, washing the cells, and removing the enzyme solution; the fifth step: adding erythrocyte lysate to lyse erythrocytes, discarding supernatant, and adding PBS to wash cell precipitate to remove erythrocyte lysate; and a sixth step: and adding NXFS glioma stem cell culture media to 6-hole cell plates coated by the lamin to perform primary culture of the glioma stem cells. The glioma stem cells with the purity of about 80% can be obtained.
Description
Technical Field
The invention relates to the technical field of stem cell culture, in particular to an in-vitro culture method and a culture medium for glioma stem cells.
Background
Malignant gliomas are the most common intracranial malignant tumors, account for about 15% of the population with overall disease, can be transformed from healthy cells in the brain and primary astrocytomas, are mostly of unknown causes and are of many types, and can be classified into astrocytomas, medulloblastomas, glioblastoma multiforme, ependymomas, oligodendrogliomas and the like according to the pathology. The tumor has no prevention method, the general survival rate after diagnosis is 12-15 months, the current effective treatment method is surgical excision treatment combined with radiotherapy and chemotherapy, however, the tumor generally grows in an infiltration type, no clear boundary exists, the postoperative recurrence rate is extremely high, and glioma is highly heterogeneous among individuals.
The difficulty of glioma in clinical research at present is that: the individual difference is not greatly researched in vivo; the difficulty of culturing glioma cells in vitro at present is as follows: the materials are difficult to obtain, the culture cost is huge, no uniform and reliable culture standard exists, the culture time is long, and the difficulty is high.
The glioma cells from patients are cultured in vitro, an in vitro malignant glioma disease model is established, the disease mechanism can be deeply researched from the perspective of cell molecules, the cells can be used in a targeted manner for screening clinical prodrug and evaluating drug effect, higher targeting performance can be realized from both research and application, and the obtained result is more accurate compared with that obtained by taking animal cells as the model.
For example, chinese patent application No. 201210299549.6 proposes a human brain glioma cell line, which has a cultivation process that reveals glioma, but the purity of glioma obtained by cultivation is not sufficient.
Therefore, there is a need to provide a culture method for glioma stem cells in vitro and a culture medium solution which can deeply study the disease mechanism.
Disclosure of Invention
The invention aims to provide a glioma stem cell in-vitro culture method with higher targeting and an optimized culture medium.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for culturing glioma stem cells in vitro by separation comprises the following steps: the method comprises the following steps:
the first step is as follows: placing a fresh glioma tissue in PBS, and shearing the glioma tissue to be minced in a sterile environment;
the second step is that: adding an Accutase solution to digest the tissue mass into a cell suspension;
the third step: adding GC glioma cell culture medium, mixing uniformly, and stopping digestion; filtering the cell suspension by a filter screen to obtain a single cell suspension;
the fourth step: adding PBS to re-suspend the precipitate, washing the cells, and removing the enzyme solution;
the fifth step: adding erythrocyte lysate to lyse erythrocytes, discarding supernatant, and adding PBS to wash cell precipitate to remove erythrocyte lysate;
And a sixth step: adding NXFS glioma stem cell culture media to a 6-hole cell plate coated by the Laminin to perform primary culture of the glioma stem cells;
wherein, the NXFS glioma stem cell culture medium comprises the following components: basal Medium, promotion Supplement, B-27, EGF, bFGF, Heparin Solution, GlutaMAX-I, Sodium sulfate, human insulin and Pen Strep.
The NXFS glioma stem cell culture medium comprises the following components by preparing 50mL of each culture medium:
NeuroCultTM-XF Basal Medium，42.3mL；
NeuroCultTM-XF Proliferation Supplement，5mL；
B-27(50x Thermofisher#17504-044)，1mL；
10ug/mL EGF，50uL；
10ug/mL bFGF，50uL；
Heparin Solution(Gibico STEMCELL#07980)，50uL；
GlutaMAX-I(100x,Gibco#35050-061)，0.5mL；
Sodium Pyrurate(100mM,Gibco#11360-070)，0.5mL；
human insulin, 25uL 0.02 mg/uL;
Pen Strep，0.5mL。
in order to achieve the purpose, the invention adopts the following technical scheme: the components of the culture medium comprise: basal Medium, promotion Supplement, B-27, EGF, bFGF, Heparin Solution, GlutaMAX-I, Sodium sulfate, human insulin and Pen Strep.
The NXFS glioma stem cell culture medium of claim 2 wherein 50mL of each formulation comprises the following components:
NeuroCultTM-XF Basal Medium，42.3mL；
NeuroCultTM-XF Proliferation Supplement，5mL；
B-27(50x Thermofisher#17504-044)，1mL；
10ug/mL EGF，50uL；
10ug/mL bFGF，50uL；
Heparin Solution(Gibico STEMCELL#07980)，50uL；
GlutaMAX-I(100x,Gibco#35050-061)，0.5mL；
Sodium Pyrurate(100mM,Gibco#11360-070)，0.5mL；
human insulin, 25uL 0.02 mg/uL;
Pen Strep，0.5mL。
compared with the prior art, the glioma stem cell in-vitro culture method and the culture medium liquid have the beneficial effects that: the glioma stem cells with the purity of about 80% can be obtained, so that the interference of mixed cells is avoided for later-stage research, a more targeted research result can be obtained, and the glioma stem cells have reference values for research and application.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:
FIG. 1 is a morphological observation (10X) of cells after passage of primary cells for the fifth passage (P5).
FIG. 2 is a morphological observation (5X) of cells after passage of primary cells for nineteenth passage (P19), showing a colony-like growth morphology.
FIG. 3 is a photograph showing immunofluorescence of glioma stem cell Nestin, Pax6, Sox2, Olig2, Vimentin, CD 44.
Fig. 4 is a diagram showing Tuj1 staining indicating that glioma stem cells have the potential to differentiate into neurons, and GFAP indicating that glioma stem cells have the potential to differentiate into astrocytes.
Fig. 5 is a statistical graph of the culture efficiency of glioma stem cells.
Detailed Description
The present invention will be described in detail below with reference to the drawings, but it should be emphasized that the following embodiments are only exemplary and are not intended to limit the scope and application of the present invention.
The invention relates to a method for culturing glioma stem cells in vitro by separation.
Example 1
Glioma stem cell specimens (female, less than 12 years of age) excised from a malignant glioblastoma.
The step of the isolation culture comprises primary culture and subculture, wherein the step of the primary culture comprises the following steps:
the first step is as follows: placing a fresh glioma tissue in PBS, and shearing the glioma tissue to be minced in a sterile environment;
specifically, the method comprises the following steps: fresh, surgically excised cytoma tissue was placed in a centrifuge tube containing 5mL PBS and placed on ice. The tumor tissue was taken out of the sterile super clean bench, placed in a dish containing PBS buffer, and the blood clots and necrotic tissue in the tumor tissue were carefully removed with forceps. The tumor tissue was cut into a paste within 5min, suspended in PBS and collected in a 15mL centrifuge tube. The working conditions were 800rpm, 3min, and then the supernatant was discarded.
The second step is that: adding Accutase solution to dissolve the tissue blocks to form cell suspension;
specifically, 2mL of Accutase solution (cell digestive juice) is added, the heavy suspension precipitation is carried out, the mixture is taken out in a water bath at 37 ℃ for 12-16 min and blown up and down once every 4min, and the shaking is carried out in the water bath until the tissue blocks are completely dissolved to form cell suspension.
The third step: adding GC glioma cell culture medium, mixing uniformly, and stopping digestion; and the cell suspension was filtered through a 70uM filter (BD) to obtain a single cell suspension.
Specifically, 8mL of serum GC glioma cell culture medium is added and blown to be uniformly mixed, and digestion is stopped; the filtration means was filtered through a 70uM strainer (BD) and centrifuged at 193g for 10min, and the supernatant was discarded. If the flocculent exists, the mixture is filtered by a 40uM filter screen.
Wherein: the GC glioma cell culture medium comprises: DMEM/F12, FBS and PS.
Specifically, each 50mL contains:
DMEM/F12(Gibco)，44.5mL；
FBS(Gibco)，5mL；
PS，0.5mL。
the fourth step: PBS was added to resuspend the pellet again, the cells were washed, and the enzyme solution was removed.
Specifically, the pellet was resuspended again with 10mL of PBS, the cells were washed, and the enzyme solution was removed as much as possible by centrifugation at 193g for 10 min.
The fifth step: adding erythrocyte lysate to lyse erythrocytes, discarding supernatant, and adding PBS to wash cell precipitate to remove erythrocyte lysate.
Specifically, 1mL of erythrocyte lysate is added, the erythrocyte is gently blown up and down for 2min, the erythrocyte is lysed, 300g is carried out for 5min, and the supernatant is discarded. The cell pellet was washed with 10mL PBS to remove red blood cell lysate. 193g, 10min centrifugation.
And a sixth step: adding PBS to suspend the cell sediment again, blowing evenly, centrifuging and discarding the supernatant.
Specifically, 10mL of PBS was added to resuspend the cell pellet, blow it evenly, and then centrifuge it to remove the supernatant.
The seventh step: and adding NXFS glioma stem cell culture media to 6-hole cell plates coated by the lamin to perform primary culture of the glioma stem cells.
Diluting Laminin with DPBS in advance and performing coating treatment;
specifically, a refrigerator-thawed Laminin (Laminin) was diluted to 10ug/mL with DPBS, coated with a six-well cell plate (Thermo Fisher #140675), and incubated at 37 ℃ for more than 2 h.
Laminin: the unopened laminin is stored at-80 deg.C. Before use, the lamin was slowly melted in a refrigerator at 4 ℃. Subpackaging with 100 uL/tube, and freezing at-20 ℃. Before use, the solution was thawed slowly in a refrigerator at 4 ℃ and then diluted to 10ug/mL with DPBS containing Ca2+/Mg2+, 1mL per 6 wells. And (4) recommending the incubation to be carried out overnight at the temperature of 2-8 ℃. Incubation was also possible for 2h at 37 ℃. Specifically, the culture medium of the NXFS glioma stem cells is 2 mL.
Wherein, the NXFS glioma stem cell culture medium comprises the following components: basal Medium, promotion Supplement, B-27, EGF, bFGF, Heparin Solution, GlutaMAX-I, Sodium sulfate, human insulin and Pen Strep.
Specifically, the preparation method of each 50mL comprises the following steps:
NeuroCultTM-XF Basal Medium，40.3mL；
NeuroCultTM-XF Proliferation Supplement，6mL；
B-27(50x Thermofisher#17504-044)，2mL；
10ug/mL EGF，50uL；
10ug/mL bFGF，50uL；
heparin Solution (Gibico STEMCELL #07980), 50 uL;
GlutaMAX-I(100x,Gibco#35050-061)，0.6mL；
Sodium Pyrurate(100mM,Gibco#11360-070)，0.6mL；
Human insulin, 25uL 0.02 mg/uL;
Pen Strep，0.5mL。
wherein, the formulation of human insulin is as follows: 0.01M HCl was filtered through 0.22uM filter and 20mg HCl was dissolved in 1.5mL ddH2In O, 37.5 uL/tube is subpackaged, and the storage temperature is-20 ℃, and the storage concentration is 13.33 ug/uL.
0.01M HCl formulation: ddH2O. high temperature Sterilization, 1.03g HCl was weighed out and then 9.5mL ddH was added2O is mixed evenly to obtain 1M HCl, 100uL is taken to be dissolved into 9.9mL ddH2O was mixed to give 0.01M HCl.
EGF (STEMCELL # 78006.1):
1mL of 0.1% BSA was added to one tube of EGF (100ug) and the tips were mixed well to a final concentration of 100 ug/mL. Continuously diluting to 10mL of 0.1% BSA with a final concentration of 10ug/mL, subpackaging, and freezing at-20 deg.C. Freeze-thaw was not allowed to exceed 3 times.
bFGF (stemgel # 78003.1):
1mL of 0.1% BSA in sterile water was added to a tube of bFGF (10ug), and the tube was mixed well with a final concentration of 10 ug/mL. Subpackaging and freezing at-20 ℃. Freeze-thaw was not allowed to exceed 3 times.
The subculture method specifically comprises the following steps: continuously replacing the NXFS glioma stem cell culture medium until the cells grow to be completely confluent, and then carrying out subculture or cryopreservation treatment.
Specifically, the old medium was aspirated and washed 1 time with PBS, Accutase digest was added, incubated at 37 ℃ until round, the side wall of the container was tapped to float at least 80% of the cells, GC glioma cell medium was added, digestion was stopped, and the cells were collected by centrifugation, after which the cells were suspended in a lamin coated culture plate with fresh NXFS glioma stem cell medium.
Referring to fig. 1, which is a morphological observation (10 ×) of glioma stem cells after the fifth passage (P5) of primary cells, the cells were overall morphologically uniform, full and few dead cells.
As shown in FIG. 2, which is a morphological observation image (5X) of cells after long-term culture (P19), the cells exhibited colony-like growth morphology and survived through 19 generations.
FIG. 3 is a photograph showing immunofluorescence of glioma stem cell Nestin, Pax6, Sox2, Olig2, Vimentin, CD 44. It can be seen that cultured glioma stem cells have neural stem cell characteristics.
Fig. 4 is a diagram showing Tuj1 staining indicating that glioma stem cells have the potential to differentiate into neurons, and GFAP indicating that glioma stem cells have the potential to differentiate into astrocytes.
Fig. 5 is a statistical graph of the culture efficiency of glioma stem cells. Specifically, in the in vitro culture of this type of cells, a single type of cells, i.e., the cultured glioma cells of the present invention, may not be obtained, and a mixture of a plurality of cell types, such as glioma cells, glioma stem cells, glial cells, neurons, etc., may be obtained. In the expected application targets (clinical research and drug development), the target cells of most interest at present are glioma stem cells, and the presence of other hybrid cells influences the subsequent research and use results, so that the culture with higher purity, namely, the existence ratio of glioma stem cells is higher is required to be obtained. The difference of the ratio is the main difference of the culture results of the current various in vitro culture methods. As shown in FIG. 5, the glioma stem cells with about 80% purity can be obtained when the generation is passaged to the 5 th generation, so that the interference of mixed cells is avoided for later-stage research, a more targeted research result can be obtained, and the glioma stem cells have reference values for research and application. In the aspect of drug development, the accuracy of drug screening can be improved, the success rate of drug development can be improved, and the condition that the in vitro test of the drug is effective and the clinical test fails can be reduced.
Example 2
Glioma stem cell specimens (female, less than 12 years of age) excised from a malignant glioblastoma.
The step of the isolation culture comprises primary culture and subculture, wherein the step of the primary culture comprises the following steps:
the first step is as follows: placing a fresh glioma tissue in PBS, and shearing the glioma tissue to be minced in a sterile environment;
specifically, the method comprises the following steps: fresh, surgically excised cytoma tissue was placed in a centrifuge tube containing 20mL PBS and placed on ice. The tumor tissue was taken out of the sterile super clean bench, placed in a dish containing PBS buffer, and the blood clots and necrotic tissue in the tumor tissue were carefully removed with forceps. The tumor tissue was cut into a paste within 5min, suspended in PBS and collected in a 15mL centrifuge tube. The working conditions were 800rpm, 3min, and then the supernatant was discarded.
The second step is that: adding Accutase solution to dissolve the tissue blocks to form cell suspension;
specifically, 4mL of Accutase solution (cell digestive juice) is added, the heavy suspension precipitation is carried out, the mixture is taken out in a water bath at 37 ℃ for 12-16 min and blown up and down once every 4min, and the mixture is shaken in the water bath until all tissue blocks are dissolved to form cell suspension.
The third step: adding GC glioma cell culture medium, mixing uniformly, and stopping digestion; and the cell suspension was filtered through a 70uM filter (BD) to obtain a single cell suspension.
Specifically, 10mL of serum GC glioma cell culture medium is added and blown to be uniformly mixed, and digestion is stopped; the filtration means was filtered through a 70uM strainer (BD) and centrifuged at 193g for 10min, and the supernatant was discarded. If the flocculent exists, the mixture is filtered by a 40uM filter screen.
Wherein: the GC glioma cell culture medium comprises: DMEM/F12, FBS and PS.
Specifically, each 50mL contains:
DMEM/F12(Gibco)，43mL；
FBS(Gibco)，6mL；
PS，1mL。
the fourth step: PBS was added to resuspend the pellet again, the cells were washed, and the enzyme solution was removed.
Specifically, the pellet was resuspended again with 20mL of PBS, the cells were washed, and the enzyme solution was removed as much as possible by centrifugation at 193g for 10 min.
The fifth step: adding erythrocyte lysate to lyse erythrocytes, discarding supernatant, and adding PBS to wash cell precipitate to remove erythrocyte lysate.
Specifically, 0.5mL of erythrocyte lysate is added, the erythrocyte is gently blown up and down for 2min, the erythrocyte is lysed, 300g is carried out for 5min, and the supernatant is discarded. The cell pellet was washed with 20mL PBS to remove red blood cell lysate. 193g, 10min centrifugation.
And a sixth step: adding PBS to suspend the cell sediment again, blowing evenly, centrifuging and discarding the supernatant.
Specifically, 20mL of PBS was added to resuspend the cell pellet, blow it evenly, and then centrifuge it to remove the supernatant.
The seventh step: and adding NXFS glioma stem cell culture media to 6-hole cell plates coated by the lamin to perform primary culture of the glioma stem cells.
Diluting Laminin with DPBS in advance and performing coating treatment;
specifically, a refrigerator-thawed Laminin (Laminin) was diluted to 20ug/mL with DPBS, coated on a six-well cell plate (Thermo Fisher #140675), and incubated at 37 ℃ for more than 2 h.
Laminin: the unopened laminin is stored at-80 deg.C. Before use, the lamin was slowly melted in a refrigerator at 4 ℃. Subpackaging with 100 uL/tube, and freezing at-20 ℃. Before use, the cells were thawed slowly in a refrigerator at 4 ℃ and then diluted to 10ug/mL with DPBS containing Ca2+/Mg2+, 2mL per 6 wells. And (4) recommending the incubation to be carried out overnight at the temperature of 2-8 ℃. Incubation was also possible for 2h at 37 ℃. Specifically, the culture medium of the NXFS glioma stem cells is 4 mL.
Wherein, the NXFS glioma stem cell culture medium comprises the following components: basal Medium, promotion Supplement, B-27, EGF, bFGF, Heparin Solution, GlutaMAX-I, Sodium sulfate, human insulin and Pen Strep.
Specifically, the preparation method of each 50mL comprises the following steps:
NeuroCultTM-XF Basal Medium，40mL；
NeuroCultTM-XF Proliferation Supplement，5.5mL；
B-27(50x Thermofisher#17504-044)，2.8mL；
10ug/mL EGF，50uL；
10ug/mL bFGF，50uL；
heparin Solution (Gibico STEMCELL #07980), 50 uL;
GlutaMAX-I(100x,Gibco#35050-061)，0.7mL；
Sodium Pyrurate(100mM,Gibco#11360-070)，0.3mL；
human insulin, 20uL 0.02 mg/uL;
Pen Strep，0.5mL。
wherein, the formulation of human insulin is as follows: 0.01M HCl was filtered through 0.22uM filter membrane, and 25mg HCl was dissolved in 1.5mL ddH2In O, 37.5 uL/tube is subpackaged, and the storage temperature is-20 ℃, and the storage concentration is 13.33 ug/uL.
0.01M HCl formulation: ddH2O. high temperature Sterilization, 1.03g HCl was weighed out and 10mL ddH was added2Mixing O to obtain 1M HCl, taking 100uL to dissolve into 12mL ddH2O was mixed to give 0.01M HCl.
EGF (STEMCELL # 78006.1):
1mL of 0.1% BSA was added to one tube of EGF (100ug) and the tips were mixed well to a final concentration of 100 ug/mL. Continuously diluting to 10mL of 0.1% BSA with a final concentration of 10ug/mL, subpackaging, and freezing at-20 deg.C. Freeze-thaw was not allowed to exceed 3 times.
bFGF (stemgel # 78003.1):
1mL of 0.1% BSA in sterile water was added to a tube of bFGF (10ug), and the tube was mixed well with a final concentration of 10 ug/mL. Subpackaging and freezing at-20 ℃. Freeze-thaw was not allowed to exceed 3 times.
The subculture method specifically comprises the following steps: continuously replacing the NXFS glioma stem cell culture medium until the cells grow to be completely confluent, and then carrying out subculture or cryopreservation treatment.
Specifically, the old medium was aspirated and washed 1 time with PBS, Accutase digest was added, incubated at 37 ℃ until round, the side wall of the container was tapped to float at least 80% of the cells, GC glioma cell medium was added, digestion was stopped, and the cells were collected by centrifugation, after which the cells were suspended in a lamin coated culture plate with fresh NXFS glioma stem cell medium.
Example 3
Glioma stem cell specimens (female, less than 12 years of age) excised from a malignant glioblastoma.
The step of the isolation culture comprises primary culture and subculture, wherein the step of the primary culture comprises the following steps:
the first step is as follows: placing a fresh glioma tissue in PBS, and shearing the glioma tissue to be minced in a sterile environment;
specifically, the method comprises the following steps: fresh, surgically excised cytoma tissue was placed in a centrifuge tube containing 20mL PBS and placed on ice. The tumor tissue was taken out of the sterile super clean bench, placed in a dish containing PBS buffer, and the blood clots and necrotic tissue in the tumor tissue were carefully removed with forceps. The tumor tissue was cut into a paste within 5min, suspended in PBS and collected in a 15mL centrifuge tube. The working conditions were 800rpm, 3min, and then the supernatant was discarded.
The second step is that: adding Accutase solution to dissolve the tissue blocks to form cell suspension;
specifically, 4mL of Accutase solution (cell digestive juice) is added, the heavy suspension precipitation is carried out, the mixture is taken out in a water bath at 37 ℃ for 12-16 min and blown up and down once every 4min, and the mixture is shaken in the water bath until all tissue blocks are dissolved to form cell suspension.
The third step: adding GC glioma cell culture medium, mixing uniformly, and stopping digestion; and the cell suspension was filtered through a 70uM filter (BD) to obtain a single cell suspension.
Specifically, 10mL of serum GC glioma cell culture medium is added and blown to be uniformly mixed, and digestion is stopped; the filtration means was filtered through a 70uM strainer (BD) and centrifuged at 193g for 10min, and the supernatant was discarded. If the flocculent exists, the mixture is filtered by a 40uM filter screen.
Wherein: the GC glioma cell culture medium comprises: DMEM/F12, FBS and PS.
Specifically, each 50mL contains:
DMEM/F12(Gibco)，45mL；
FBS(Gibco)，4mL；
PS，1mL。
the fourth step: PBS was added to resuspend the pellet again, the cells were washed, and the enzyme solution was removed.
Specifically, the pellet was resuspended again with 10mL of PBS, the cells were washed, and the enzyme solution was removed as much as possible by centrifugation at 193g for 10 min.
The fifth step: adding erythrocyte lysate to lyse erythrocytes, discarding supernatant, and adding PBS to wash cell precipitate to remove erythrocyte lysate.
Specifically, 0.5mL of erythrocyte lysate is added, the erythrocyte is gently blown up and down for 2min, the erythrocyte is lysed, 300g is carried out for 5min, and the supernatant is discarded. The cell pellet was washed with 20mL PBS to remove red blood cell lysate. 193g, 10min centrifugation.
And a sixth step: adding PBS to suspend the cell sediment again, blowing evenly, centrifuging and discarding the supernatant.
Specifically, 10mL of PBS was added to resuspend the cell pellet, blow it evenly, and then centrifuge it to remove the supernatant.
The seventh step: and adding NXFS glioma stem cell culture media to 6-hole cell plates coated by the lamin to perform primary culture of the glioma stem cells.
Diluting Laminin with DPBS in advance and performing coating treatment;
specifically, a refrigerator-thawed Laminin (Laminin) was diluted to 20ug/mL with DPBS, coated on a six-well cell plate (Thermo Fisher #140675), and incubated at 37 ℃ for more than 2 h.
Laminin: the unopened laminin is stored at-80 deg.C. Before use, the lamin was slowly melted in a refrigerator at 4 ℃. Subpackaging with 100 uL/tube, and freezing at-20 ℃. Before use, the cells were thawed slowly in a refrigerator at 4 ℃ and then diluted to 10ug/mL with DPBS containing Ca2+/Mg2+, 2mL per 6 wells. And (4) recommending the incubation to be carried out overnight at the temperature of 2-8 ℃. Incubation was also possible for 2h at 37 ℃. Specifically, the culture medium of the NXFS glioma stem cells is 4 mL.
Wherein, the NXFS glioma stem cell culture medium comprises the following components: basal Medium, promotion Supplement, B-27, EGF, bFGF, Heparin Solution, GlutaMAX-I, Sodium sulfate, human insulin and Pen Strep.
Specifically, the preparation method of each 50mL comprises the following steps:
NeuroCultTM-XF Basal Medium，44mL；
NeuroCultTM-XF Proliferation Supplement，4.5mL；
B-27(50x Thermofisher#17504-044)，1.8mL；
10ug/mL EGF，50uL；
10ug/mL bFGF，50uL；
heparin Solution (Gibico STEMCELL #07980), 50 uL;
GlutaMAX-I(100x,Gibco#35050-061)，0.7mL；
Sodium Pyrurate(100mM,Gibco#11360-070)，0.3mL；
human insulin, 20uL 0.02 mg/uL;
Pen Strep，0.5mL。
wherein, the formulation of human insulin is as follows: 0.01M HCl was filtered through 0.22uM filter membrane, and 25mg HCl was dissolved in 1.5mL ddH2In O, 37.5 uL/tube is subpackaged, and the storage temperature is-20 ℃, and the storage concentration is 13.33 ug/uL.
0.01M HCl formulation: ddH2O. high temperature Sterilization, 1.03g HCl was weighed out and 10mL ddH was added2Mixing O to obtain 1M HCl, taking 100uL to dissolve into 12mL ddH2O was mixed to give 0.01M HCl.
EGF (STEMCELL # 78006.1):
1mL of 0.1% BSA was added to one tube of EGF (100ug) and the tips were mixed well to a final concentration of 100 ug/mL. Continuously diluting to 10mL of 0.1% BSA with a final concentration of 10ug/mL, subpackaging, and freezing at-20 deg.C. Freeze-thaw was not allowed to exceed 3 times.
bFGF (stemgel # 78003.1):
1mL of 0.1% BSA in sterile water was added to a tube of bFGF (10ug), and the tube was mixed well with a final concentration of 10 ug/mL. Subpackaging and freezing at-20 ℃. Freeze-thaw was not allowed to exceed 3 times.
The subculture method specifically comprises the following steps: continuously replacing the NXFS glioma stem cell culture medium until the cells grow to be completely confluent, and then carrying out subculture or cryopreservation treatment.
Specifically, the old medium was aspirated and washed 1 time with PBS, Accutase digest was added, incubated at 37 ℃ until round, the side wall of the container was tapped to float at least 80% of the cells, GC glioma cell medium was added, digestion was stopped, and the cells were collected by centrifugation, after which the cells were suspended in a lamin coated culture plate with fresh NXFS glioma stem cell medium.
Through the multiple embodiments, the culture method disclosed by the invention can obtain the glioma stem cells with the purity of about 80% already when the culture method is passaged to the 5 th generation, so that the interference of mixed cells is avoided for later-stage research, a more targeted research result can be obtained, and the culture method has higher research and application reference values. In the aspect of drug development, the accuracy of drug screening can be improved, the success rate of drug development can be improved, and the condition that the in vitro test of the drug is effective and the clinical test fails can be reduced.
Of course, those skilled in the art will recognize that the above-described embodiments are illustrative only, and not intended to be limiting, and that changes and modifications may be made thereto without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (4)
1. A glioma stem cell in-vitro isolated culture method is characterized by comprising the following steps:
the first step is as follows: placing a fresh glioma tissue in PBS, and shearing the glioma tissue to be minced in a sterile environment;
the second step is that: adding an Accutase solution to digest the tissue mass into a cell suspension;
the third step: adding GC glioma cell culture medium, mixing uniformly, and stopping digestion; filtering the cell suspension by a filter screen to obtain a single cell suspension;
the fourth step: adding PBS to re-suspend the precipitate, washing the cells, and removing the enzyme solution;
the fifth step: adding erythrocyte lysate to lyse erythrocytes, discarding supernatant, and adding PBS to wash cell precipitate to remove erythrocyte lysate;
and a sixth step: adding NXFS glioma stem cell culture media to a 6-hole cell plate coated by the Laminin to perform primary culture of the glioma stem cells;
wherein the NXFS glioma stem cell culture medium comprises the following components: basal Medium, promotion Supplement, B-27, EGF, bFGF, Heparin Solution, GlutaMAX-I, Sodium sulfate, human insulin and Pen Strep.
2. The method for isolated culture of glioma stem cells in vitro of claim 1, wherein the NXFS glioma stem cell culture medium comprises, per 50mL of preparation:
NeuroCultTM-XF Basal Medium，40-42.3mL；
NeuroCultTM-XF Proliferation Supplement，5-6mL；
B-27(50x Thermofisher#17504-044)，1-2.8mL；
10ug/mL EGF，50uL；
10ug/mL bFGF，50uL；
Heparin Solution(Gibico STEMCELL#07980)，50uL；
GlutaMAX-I(100x,Gibco#35050-061)，0.5-0.7mL；
Sodium Pyrurate(100mM,Gibco#11360-070)，0.3-0.6mL；
human insulin, 25uL 0.02 mg/uL;
Pen Strep，0.5mL。
3. an NXFS glioma stem cell culture medium comprising: basal Medium, promotion Supplement, B-27, EGF, bFGF, Heparin Solution, GlutaMAX-I, Sodium sulfate, human insulin and Pen Strep.
4. The NXFS glioma stem cell culture medium of claim 3 wherein 50mL of the NXFS glioma stem cell culture medium is prepared with the following components:
NeuroCultTM-XF Basal Medium，40-42.3mL；
NeuroCultTM-XF Proliferation Supplement，6、5-6mL；
B-27(50x Thermofisher#17504-044)，1-2.8mL；
10ug/mL EGF，50uL；
10ug/mL bFGF，50uL；
Heparin Solution(Gibico STEMCELL#07980)，50uL；
GlutaMAX-I(100x,Gibco#35050-061)，0.5-0.7mL；
Sodium Pyrurate(100mM,Gibco#11360-070)，0.3-0.6mL；
human insulin, 25uL 0.02 mg/uL;
Pen Strep，0.5mL。
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