CN114480285B - Glioma organoid resuscitating liquid, culture liquid and resuscitating culture method - Google Patents
Glioma organoid resuscitating liquid, culture liquid and resuscitating culture method Download PDFInfo
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Abstract
The invention provides a glioma organoid resuscitation liquid, a culture liquid and a resuscitation culture method, belonging to the technical field of glioma organoid resuscitation. The resuscitation fluid comprises taxifolin and calycosin. The glioma organoid resuscitation fluid can greatly reduce the damage of DMSO to glioma organoids and the damage of freezing to glioma organoids when the glioma organoids are resuscitated, can remarkably improve the vitality of glioma organoids and improve the survival rate of glioma organoids after being resuscitated; and the animal serum and heterogeneous source proteins are not contained, and the pollution of viruses, bacteria, mycoplasma and the like can be avoided.
Description
Technical Field
The invention belongs to the technical field of glioma organoid resuscitation, and particularly relates to glioma organoid resuscitation liquid, culture liquid and a resuscitation culture method.
Background
Gliomas are common malignant tumors of the central nervous system, often grow in an infiltrative growth mode, are not clearly separated from surrounding brain tissues, have high difficulty in complete excision and are easy to relapse after operation, so that the gliomas are particularly important for in vitro research.
Organoid studies are a recent technique for in vitro cell culture. The organoid model can well simulate the microenvironment of cells in vivo, and has great advantages for constructing a research model with physiological functions in vitro. The in vitro culture of glioma organoids adopts a special 3D culture mode, but the freezing method is a conventional cell freezing method. The freezing and preserving of the cells in the laboratory are mostly carried out by mixing dimethyl sulfoxide, bovine serum, culture solution and the like according to a certain proportion, however, the dimethyl sulfoxide has high toxicity, and the toxicity can poison and damage the cells to a certain extent, so that the activity of the cells is reduced, and the damage to glioma organoids is more obvious. Therefore, in the recovery of glioma organoids, a reagent is needed to improve the viability of glioma organoids and increase the survival rate.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide the glioma organoid resuscitation fluid, and when the glioma organoids are resuscitated, the resuscitation fluid is added into the culture fluid, so that the vitality and the activity rate of the glioma organoids can be improved, and the proliferation speed of the glioma organoids can be accelerated.
The second object of the invention is to provide a glioma organoid complex culture solution which can improve the activity and the activity rate of glioma organoids and accelerate the proliferation speed of glioma organoids.
The third object of the invention is to provide a recovery culture method of glioma-like devices.
The invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided a glioma organoid resuscitation fluid comprising taxifolin and calycosin.
Further, the glioma organoid resuscitation fluid mainly comprises taxifolin with an effective concentration range of 0.1-10 mug/ml and calycosin with an effective concentration range of 1-50 mug/ml.
In a second aspect of the invention, there is provided a glioma organoid resuscitation fluid comprising the glioma organoid resuscitation fluid.
Further, the preparation method of the glioma organoid resuscitation culture solution comprises the following steps:
(1) Preparing 1000 times concentrated glioma organoid resuscitation fluid mother liquor: accurately weighing taxifolin and calycosin in a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and gently shaking the centrifuge tube until the taxifolin and calycosin are dissolved, wherein mother liquor contains 0.1-10mg/ml taxifolin and 1-50mg/ml calycosin;
(2) And (3) filtering and sterilizing: the prepared mother solution is transferred into a laboratory through a material channel and is filtered and sterilized by a 0.22 mu m filter in a biosafety cabinet;
(3) Preparing a resuscitating culture solution: taking preheated glioma organoid growth culture solution, diluting the filtered sterilized recovery solution mother solution 1000 times according to the volume of the culture solution, and uniformly mixing for later use.
Further, the glioma organoid growth medium comprises DMEM high-glucose cell culture medium, 1 XB 27 serum replacement, 20ng/mL EGF, 10mM niacinamide, 10. Mu.M ascorbic acid, 25ng/mL FGF10, 5. Mu. M A83-01, 25ng/mL R-spondin-1.
In a third aspect of the present invention, a method for recovering and culturing glioma organoids is provided, wherein the method comprises the steps of:
(1) Melting matrigel at 4deg.C in advance, placing on ice before experiment, pre-cooling gun head at-20deg.C in advance, and preheating culture dish in incubator in advance;
(2) Taking out the frozen glioma cells, immediately transferring the glioma cells into a water bath kettle at 37 ℃ for thawing, and shaking while thawing;
(3) Adding at least 5ml of precooled PBS into a centrifuge tube, adding the thawed cell suspension into the centrifuge tube, lightly mixing and centrifuging;
(4) Removing the supernatant, adding a proper amount of glioma organoid growth culture solution, and lightly suspending cells;
(5) Mixing the cell suspension and matrigel in a ratio of 1:5, dripping the mixture into a culture dish after uniformly mixing, taking care of not generating bubbles, standing for 3min, and putting the culture dish into an incubator for about 15min to solidify the matrigel;
(6) Taking out the culture dish, adding the prepared resuscitating culture solution into the culture dish, and placing the culture dish into an incubator for culturing;
(7) After 48 hours of culture, the glioma organoid growth medium is changed for continuous culture.
Compared with the prior art, the invention has the beneficial effects that:
the glioma organoid resuscitation fluid provided by the invention can greatly reduce the damage of DMSO to glioma organoids and the damage of freezing to glioma organoids when the glioma organoids are resuscitated, can obviously improve the vitality of glioma organoids and improve the survival rate of glioma organoids after resuscitating.
The glioma organoid resuscitation fluid provided by the invention does not contain animal serum and heterogeneous source proteins, and can avoid pollution of viruses, bacteria, mycoplasma and the like.
Drawings
FIG. 1 is a CCK8 experimental data analysis of taxifolin concentration optimization;
FIG. 2 is a graph showing analysis of CCK8 experimental data for optimizing Calycosin concentration;
FIG. 3 is an analysis of experimental CCK8 data for different concentration drug combinations;
FIG. 4 shows the results of immunohistochemical staining.
Detailed Description
The invention is described in further detail below with reference to the attached drawing figures:
The invention provides glioma organoid resuscitation fluid, which mainly comprises taxifolin and calycosin.
As a possible scheme, the glioma organoid resuscitation fluid mainly comprises taxifolin with an effective concentration range of 0.1-10 mug/ml and calycosin with a concentration range of 1-50 mug/ml.
The cell is cultured by adopting a culture solution added with a resuscitating solution, wherein the resuscitating solution comprises taxifolin and calycosin, and the effective concentration range refers to the final use concentration of taxifolin and calycosin in the culture solution.
The taxifolin is a dihydroflavonol compound, belongs to vitamin P group, is a widely applied bioactive agent, and has various biological activities in human body, including antioxidation, free radical removal and the like, as with other flavonoid compounds; in particular, the presence of 4,7-OH and 3',4' -ortho-dihydroxyl thereof makes the antioxidant capacity of the bioflavonoids superior to that of general bioflavonoids. Taxifolin is identified as one of the more potent vitamin P that guarantees glioma organoids to survive. The effective concentration range of taxifolin is 0.1-10 mug/ml.
The calycosin has pharmacological effects of enhancing immunity, enhancing antioxidant capacity, protecting glioma organoids, and promoting glioma organoids survival. The effective concentration range of the calycosin is 1-50 mug/ml.
The invention also provides a glioma organoid resuscitation medium, which comprises the glioma organoid resuscitation medium.
The preparation method of the glioma organoid resuscitation culture solution comprises the following steps:
(1) Preparing 1000 times concentrated glioma organoid resuscitation fluid mother liquor: accurately weighing taxifolin and calycosin in a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and gently shaking the centrifuge tube until the taxifolin and calycosin are dissolved, wherein mother liquor contains 0.1-10mg/ml taxifolin and 1-50mg/ml calycosin;
(2) And (3) filtering and sterilizing: the prepared mother solution is transferred into a laboratory through a material channel and is filtered and sterilized by a 0.22 mu m filter in a biosafety cabinet;
(3) Preparing a resuscitating culture solution, namely taking a preheated glioma organoid growth culture solution (the glioma organoid growth culture solution comprises DMEM high sugar cell culture solution, 1 XB 27 serum substitute, 20ng/mL EGF, 10mM niacinamide, 10 mu M ascorbic acid, 25ng/mL FGF10, 5 mu M A-01 and 25ng/mL R-spondin-1), diluting the sterilized resuscitating solution mother liquor 1000 times according to the volume of the culture solution (meaning diluting the sterilized resuscitating solution mother liquor 1000 times by the glioma organoid growth culture solution), and uniformly mixing for later use.
The invention also provides a recovery culture method of the glioma organ, which utilizes the glioma organ recovery culture solution to recover the frozen glioma organ, and comprises the following steps:
(1) Melting matrigel at 4deg.C in advance, placing on ice before experiment, pre-cooling gun head at-20deg.C in advance, and preheating culture dish in incubator in advance;
(2) Taking out the frozen glioma cells, immediately transferring the glioma cells into a water bath kettle at 37 ℃ for thawing, and shaking while thawing;
(3) Adding at least 5ml of precooled PBS into a centrifuge tube, adding the thawed cell suspension into the centrifuge tube, and centrifuging (1000 rpm,5 min) after gently mixing;
(4) Removing the supernatant, adding a proper amount of glioma organoid growth culture broth (glioma organoid growth culture broth comprises DMEM high sugar cell culture broth, 1 XB 27 serum substitute, 20ng/mL EGF, 10mM niacinamide, 10 μM ascorbic acid, 25ng/mL FGF10, 5 μ M A83-01, 25ng/mL R-spondin-1), and lightly suspending the cells;
(5) Mixing the cell suspension and matrigel in a ratio of 1:5, dripping the mixture into a culture dish after uniformly mixing, taking care of not generating bubbles, standing for 3min, and putting the culture dish into an incubator for about 15min to solidify the matrigel;
(6) Taking out the culture dish, adding the prepared resuscitating culture solution into the culture dish, and placing the culture dish into a culture box with the temperature of 37 ℃ and the concentration of 5% CO 2 for culture;
(7) After 48 hours of culture, the glioma organoid growth medium is changed for continuous culture.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental reagents used, without any particular instruction, were purchased from conventional reagent manufacturers.
The experimental environment, experimental materials and instrument equipment required to be prompted and described in the invention are as follows:
1. Experimental environment: and (3) operating in a biological safety cabinet in a laboratory in a GMP environment.
2. Experimental reagent: calycosin, taxifolin, cell culture solution, phosphate buffer PBS (PBS is an existing buffer solution, and is not described here).
3. Instrument and apparatus: the device comprises a CO 2 incubator, a biosafety cabinet, an enzyme-labeling instrument, a centrifuge, an electronic balance and a 96-well plate.
Example 1
A glioma organoid resuscitation fluid mainly comprises taxifolin with effective concentration range of 0.1-10 μg/ml.
Specifically, in this example, the glioma organoid resuscitation fluid contains taxifolin at a concentration of 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml.
Specifically, in this embodiment, the cells are glioma cells.
The method specifically comprises the following steps:
(1) Melting matrigel at 4deg.C, placing on ice before experiment, pre-cooling gun head at-20deg.C, and preheating culture dish in incubator.
(2) Preparing taxifolin mother liquor: and precisely weighing taxifolin into a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and gently shaking the centrifuge tube until taxifolin is dissolved. ( The concentration of taxifolin mother liquor is as follows: 0.1mg/ml, 0.5mg/ml, 1mg/ml, 5mg/ml, 10mg/ml )
(3) And (3) filtering and sterilizing: the prepared taxifolin mother liquor is transferred into a laboratory through a material channel and filtered and sterilized in a biosafety cabinet by a 0.22 mu m filter.
(4) Preparing a pre-use recovery culture solution, taking a pre-heated glioma organoid growth culture solution, diluting the taxifolin mother solution by 1000 times according to the volume of the culture solution, and uniformly mixing for later use.
(5) Taking out the frozen glioma cells, immediately transferring into a water bath kettle at 37 ℃ for thawing, and shaking while thawing.
(6) At least 5ml of pre-chilled PBS was added to the centrifuge tube, and the thawed cell suspension was added to the centrifuge tube, gently mixed and centrifuged (1000 rpm,5 min).
(7) The supernatant was discarded, and appropriate amount of glioma organoid growth medium was added, and the cells were gently suspended.
(8) Mixing the cell suspension and matrigel at a volume ratio of 1:5, dripping the mixture into a culture dish after uniformly mixing, taking care of not generating bubbles, standing for 3min, and placing the culture dish into an incubator for about 15min to solidify the matrigel.
(9) The culture dish was taken out, and a proper amount of the resuscitating culture solution prepared in step 4 was added to the culture dish, and the mixture was placed in an incubator at 37℃with 5% CO 2.
(10) After culturing for 48 hours, the glioma organoid growth culture solution is changed for continuous culture.
(11) After culturing glioma organoid growth medium for 24 hours, the medium is discarded, washed twice with PBS, matrigel lytic enzyme is added, and the culture medium is put on ice for digestion for 1-2 hours, then transferred into a 15ml centrifuge tube, centrifuged for 5 minutes at 1000rpm, and washed once with PBS.
(12) After centrifugation, the supernatant was discarded, and the glioma organoid growth medium was added to suspend the cells, which were transferred to 96-well plates with 100 μl of cell suspension per well.
(13) Mu.l of CCK8 reagent was added to each well, and the mixture was placed in an incubator at 37℃for 2 hours.
(14) The absorbance at 450nm was measured with a microplate reader.
Example two
A glioma organoid resuscitation fluid mainly comprises calycosin with effective concentration range of 1-50 μg/ml.
Specifically, in this example, the glioma organoid resuscitation fluid contains calycosin at a concentration of 1 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml.
Specifically, in this embodiment, the cells are glioma cells.
The method specifically comprises the following steps:
(1) Melting matrigel at 4deg.C, placing on ice before experiment, pre-cooling gun head at-20deg.C, and preheating culture dish in incubator.
(2) Preparing a calycosin mother solution: accurately weighing calycosin in a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and gently shaking the centrifuge tube until the calycosin is dissolved. ( The concentration of the calycosin mother solution is as follows: 1mg/ml, 5mg/ml, 10mg/ml, 20mg/ml, 50mg/ml )
(3) And (3) filtering and sterilizing: the stock solution of calycosin prepared above was passed through a material channel into a laboratory and sterilized by filtration through a 0.22 μm filter in a biosafety cabinet.
(4) Preparing a pre-use recovery culture solution, taking a pre-heated glioma organoid growth culture solution, diluting the calycosin mother solution by 1000 times according to the volume of the culture solution, and uniformly mixing for later use.
(5) Taking out the frozen glioma cells, immediately transferring into a water bath kettle at 37 ℃ for thawing, and shaking while thawing.
(6) At least 5ml of pre-chilled PBS or glioma organoid growth medium was added to the centrifuge tube, and the thawed cell suspension was added to the centrifuge tube, gently mixed and centrifuged (1000 rpm,5 min).
(7) The supernatant was discarded, and appropriate amount of glioma organoid growth medium was added, and the cells were gently suspended.
(8) Mixing the cell suspension with matrigel at a ratio of 1:5, dripping the mixture into a culture dish after mixing, taking care of not generating bubbles, standing for 3min, and placing the culture dish into an incubator for about 15min to solidify the matrigel.
(9) The culture dish was taken out, and an appropriate amount of the culture solution prepared in step 4 was added to the culture dish, and the mixture was placed in an incubator at 37℃with 5% CO 2.
(10) After culturing for 48 hours, the glioma organoid growth culture solution is changed for continuous culture.
(11) After culturing glioma organoid growth medium for 24 hours, the medium is discarded, washed twice with PBS, matrigel lytic enzyme is added, and the culture medium is put on ice for digestion for 1-2 hours, then transferred into a 15ml centrifuge tube, centrifuged for 5 minutes at 1000rpm, and washed once with PBS.
(12) After centrifugation, the supernatant was discarded, and the glioma organoid growth medium was added to suspend the cells, which were transferred to 96-well plates with 100 μl of cell suspension per well.
(13) Mu.l of CCK8 reagent was added to each well, and the mixture was placed in an incubator at 37℃for 2 hours.
(14) The absorbance at 450nm was measured with a microplate reader.
Example III
A glioma organoid resuscitation fluid mainly comprises taxifolin with effective concentration range of 0.1-10mg/ml, and calycosin with effective concentration range of 1-50 mg/ml.
Specifically, in this example, the glioma organoid resuscitation fluid contained 5 μg/ml taxifolin and 1 μg/ml calycosin, 5 μg/ml taxifolin and 10 μg/ml calycosin, 5 μg/ml taxifolin and 50 μg/ml calycosin, 0.1 μg/ml taxifolin and 10 μg/ml calycosin, 1 μg/ml taxifolin and 10 μg/ml calycosin, 10 μg/ml taxifolin and 10 μg/ml calycosin.
Specifically, in this embodiment, the cells are glioma cells.
The method specifically comprises the following steps:
(1) Melting matrigel at 4deg.C, placing on ice before experiment, pre-cooling gun head at-20deg.C, and preheating culture dish in incubator.
(2) Preparing glioma organoid resuscitation fluid mother liquor: accurately weighing taxifolin and calycosin by using an analytical balance, adding a proper amount of sterile water into a centrifuge tube, and gently shaking the centrifuge tube until the taxifolin and calycosin are dissolved. ( The concentrations of taxifolin and calycosin in the glioma organoid resuscitation fluid mother liquor are respectively as follows: 5mg/ml and 1mg/ml, 5mg/ml and 10mg/ml, 5mg/ml and 50mg/ml, 0.1mg/ml and 10mg/ml, 1mg/ml and 10mg/ml, 10mg/ml and 10mg/ml )
(3) And (3) filtering and sterilizing: the prepared glioma organoid resuscitation fluid mother liquor is transmitted into a laboratory through a material channel, and is filtered and sterilized by a 0.22 mu m filter in a biosafety cabinet.
(4) Preparing a pre-use recovery culture solution, taking a pre-heated glioma organoid growth culture solution, diluting the glioma organoid recovery solution mother solution 1000 times according to the volume of the culture solution, and uniformly mixing for later use.
(5) Taking out the frozen glioma cells, immediately transferring into a water bath kettle at 37 ℃ for thawing, and shaking while thawing.
(6) At least 5ml of pre-chilled PBS was added to the centrifuge tube, and the thawed cell suspension was added to the centrifuge tube, gently mixed and centrifuged (1000 rpm,5 min).
(7) The supernatant was discarded, and appropriate amount of glioma organoid growth medium was added, and the cells were gently suspended.
(8) Mixing the cell suspension with matrigel at a ratio of 1:5, dripping the mixture into a culture dish after mixing, taking care of not generating bubbles, standing for 3min, and placing the culture dish into an incubator for about 15min to solidify the matrigel.
(9) The culture dish was taken out, and a proper amount of the resuscitating culture solution prepared in step 4 was added to the culture dish, and the mixture was placed in an incubator at 37℃with 5% CO 2.
(10) After culturing for 48 hours, the glioma organoid growth culture solution is changed for continuous culture.
(11) After culturing glioma organoid growth medium for 24 hours, the medium is discarded, washed twice with PBS, matrigel lytic enzyme is added, and the culture medium is put on ice for digestion for 1-2 hours, then transferred into a 15ml centrifuge tube, centrifuged for 5 minutes at 1000rpm, and washed once with PBS.
(12) After centrifugation, the supernatant was discarded, and the glioma organoid growth medium was added to suspend the cells, which were transferred to 96-well plates with 100 μl of cell suspension per well.
(13) Mu.l of CCK8 reagent was added to each well, and the mixture was placed in an incubator at 37℃for 2 hours.
(14) The absorbance at 450nm was measured with a microplate reader.
Comparative example
A glioma organoid resuscitation fluid mainly comprises glioma organoid growth culture fluid, does not contain taxifolin, and does not contain calycosin.
Specifically, in this embodiment, the cells are glioma cells.
The method specifically comprises the following steps:
(1) Melting matrigel at 4deg.C, placing on ice before experiment, pre-cooling gun head at-20deg.C, and preheating culture dish in incubator.
(2) Taking out the frozen glioma cells, immediately transferring into a water bath kettle at 37 ℃ for thawing, and shaking while thawing.
(3) At least 5ml of pre-chilled PBS or glioma organoid growth medium was added to the centrifuge tube, and the thawed cell suspension was added to the centrifuge tube, gently mixed and centrifuged (1000 rpm,5 min).
(4) The supernatant was discarded, and appropriate amount of glioma organoid growth medium was added, and the cells were gently suspended.
(5) Mixing the cell suspension with matrigel at a ratio of 1:5, dripping the mixture into a culture dish after mixing, taking care of not generating bubbles, standing for 3min, and placing the culture dish into an incubator for about 15min to solidify the matrigel.
(6) The culture dish is taken out, a proper amount of glioma organoid growth culture solution is added into the culture dish, and the culture dish is placed into an incubator with the temperature of 37 ℃ and the concentration of 5% CO 2.
(7) After culturing for 48 hours, the glioma organoid growth culture solution is changed for continuous culture.
(8) After further incubation for 24 hours, the broth was discarded, washed twice with PBS, matrigel lytic enzyme was added, and digested on ice for 1-2 hours, then transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 5min, and washed once with PBS.
(9) After centrifugation, the supernatant was discarded, and the glioma organoid growth medium was added to suspend the cells, which were transferred to 96-well plates with 100 μl of cell suspension per well.
(10) Mu.l of CCK8 reagent was added to each well, and the mixture was placed in an incubator at 37℃for 2 hours.
(11) The absorbance at 450nm was measured with a microplate reader.
The results of the comparison between the first example and the control example are shown in FIG. 1, and Table 1 shows CCK8 experimental data for optimization of taxifolin concentration.
TABLE 1
As can be seen from FIG. 1 and Table 1, when the glioma cells were resuscitated, taxifolin was added to the culture solution at different concentrations, and as the concentration increased, the cell proliferation activity was stronger, and as the concentration increased, the cell proliferation activity was strongest, and as the concentration was 5 μg/ml, the cell proliferation activity was in a flattened trend in the range of 5 μg/ml to 10 μg/ml, so that 5 μg/ml of taxifolin was selected as the optimal concentration.
The results of comparison of example two with the control are shown in FIG. 2, and Table 2 shows CCK8 experimental data for optimization of taxifolin concentration.
As shown in FIG. 2 and Table 2, when the glioma cells were resuscitated, calycosin was added to the culture solution at various concentrations, and as the concentration increased, the cell proliferation activity was stronger in the range of 0. Mu.g/ml to 10. Mu.g/ml, and as the concentration increased, the cell proliferation activity was strongest in the range of 10. Mu.g/ml to 50. Mu.g/ml, and the cell proliferation activity was decreased in the range of 10. Mu.g/ml, so that the optimum concentration of Calycosin was selected as 10. Mu.g/ml.
TABLE 2
Example three was compared with the control and the results are shown in FIG. 3, and Table 3 shows the CCK8 experimental data for optimization of taxifolin concentration.
TABLE 3 Table 3
As shown in the figures 3 and the table 3, when glioma cells are resuscitated, the culture solution is added with the taxifolin and the calycosin with different concentrations, and the results show that the combination of the different concentrations can improve the proliferation activity of the cells to different degrees, wherein the combination of the taxifolin with 5 mug/ml and the calycosin with 10 mug/ml is particularly remarkable in improving the proliferation activity of the cells, and therefore, the cell activity enhancing solution adopts the taxifolin with 5 mug/ml and the calycosin with 10 mug/ml as the optimal conditions.
Blank examples
A glioma organoid resuscitation fluid mainly comprises taxifolin with effective concentration range of 0.1-10 μg/ml, and calycosin with effective concentration range of 1-50 μg/ml.
Specifically, in this example, the glioma organoid resuscitation fluid does not include taxifolin, and does not include calycosin.
(1) Cell culture broth was taken and added to 96-well plates at 100 μl per well and placed in an incubator at 37deg.C with 5% CO 2.
(2) After 48 hours of culture, the glioma organoid growth medium is changed for continuous culture.
(3) After 24 hours, the culture medium was discarded, 100. Mu.l of the culture medium was added to each well, and 10. Mu.l of CCK8 reagent was added thereto, and the mixture was placed in an incubator at 37℃for 2 hours.
(4) The absorbance at 450nm was measured with a microplate reader.
Table 4 shows the CCK8 experimental data for the blank group.
TABLE 4 Table 4
| Group of | Blank parallel 1 | Blank parallel 2 | Blank parallel 3 | Blank parallel 4 |
| OD value | 0.1744 | 0.1680 | 0.1722 | 0.1698 |
The data obtained in the blank examples are mainly used for eliminating background absorbance interference of glioma organoid growth culture fluid in blank holes when calculating the absorbance of the first, second, third and comparison examples, and subtracting the absorbance interference in calculation.
Example four morphological and immunofluorescence identification of glioma primary cells
A glioma organoid resuscitation fluid mainly comprises taxifolin with effective concentration range of 0.1-10 μg/ml, and calycosin with effective concentration range of 1-50 μg/ml.
Specifically, in this example, the glioma organoid resuscitation fluid mainly contains taxifolin and Calycosin at an effective concentration of 5 μg/ml and 10 μg/ml.
Specifically, in this embodiment, the cells are primary glioma cells.
Specifically, in this example, the primary antibody is a rabbit anti-human (SOX 2) antibody, a rabbit anti-human (Ki-67) antibody, and the secondary antibody is a goat anti-rabbit antibody.
The method specifically comprises the following steps:
(1) Melting matrigel at 4deg.C, placing on ice before experiment, pre-cooling gun head at-20deg.C, and preheating culture dish in incubator.
(2) Preparing glioma organoid resuscitation fluid mother liquor: accurately weighing taxifolin and calycosin by using an analytical balance, adding a proper amount of sterile water into a centrifuge tube, and gently shaking the centrifuge tube until the taxifolin and calycosin are dissolved. ( The concentration of taxifolin and calycosin in the mother solution of glioma organoid resuscitation fluid is as follows: 5mg/ml and 10mg/ml )
(3) And (3) filtering and sterilizing: the prepared glioma organoid resuscitation fluid mother liquor is transmitted into a laboratory through a material channel, and is filtered and sterilized by a 0.22 mu m filter in a biosafety cabinet.
(4) Preparing a pre-use recovery culture solution, taking a pre-heated glioma organoid growth culture solution, diluting the glioma organoid recovery solution mother solution 1000 times according to the volume of the culture solution, and uniformly mixing for later use.
(5) Taking out the frozen glioma cells, immediately transferring into a water bath kettle at 37 ℃ for thawing, and shaking while thawing.
(6) At least 5ml of pre-chilled PBS was added to the centrifuge tube, and the thawed cell suspension was added to the centrifuge tube, gently mixed and centrifuged (1000 rpm,5 min).
(7) The supernatant was discarded, and appropriate amount of glioma organoid growth medium was added, and the cells were gently suspended.
(8) Mixing the cell suspension with matrigel at a ratio of 1:5, dripping the mixture into a culture dish after mixing, taking care of not generating bubbles, standing for 3min, and placing the culture dish into an incubator for about 15min to solidify the matrigel.
(9) The culture dish was taken out, and a proper amount of the resuscitating culture solution prepared in step 4 was added to the culture dish, and the mixture was placed in an incubator at 37℃with 5% CO 2.
(10) After culturing for 48 hours, the glioma organoid growth culture solution is changed for continuous culture.
(11) Culturing for three weeks until glioma organoids grow to 2mm.
(12) Paraffin-embedded sections were prepared.
(13) Baking slices: taking paraffin embedded slices, and baking the slices in an oven for 2 hours.
(14) Paraffin-embedded sections were dewaxed and hydrated: sequentially placing the slices into xylene I15 min-xylene II 15 min-xylene III 15 min-absolute ethanol I5 min-absolute ethanol II 5min-85% alcohol 5min-75% alcohol 5 min-distilled water for washing.
(15) Antigen retrieval: the tissue slice is placed in a repair box filled with citric acid antigen repair buffer solution (pH6.0) for antigen repair in a microwave oven, the medium fire is 8min to boiling, the fire is stopped for 8min, the heat preservation is carried out, the medium fire is changed to the low fire for 7min, and excessive evaporation of the buffer solution is prevented in the process, and the slice is not dried. After natural cooling, the slide was washed with PBS (pH 7.4) with shaking on a decolorizing shaker for 3 times, 5min each.
(16) Blocking endogenous peroxidases: the sections were incubated in 3% hydrogen peroxide solution at room temperature for 25min in the absence of light, and the slides were washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each.
(17) Serum blocking, namely 3% BSA is dripped into a histochemical ring to uniformly cover tissues, and the tissue is blocked for 30min at room temperature.
(18) Adding an antibody: the blocking solution is gently thrown away, PBS is dripped on the slice, the slice is horizontally placed in a wet box for incubation at 4 ℃ for overnight. (A small amount of water was added to the wet box to prevent evaporation of antibody)
(19) Adding a secondary antibody: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (3) dripping secondary antibodies (Alexa 667 marked) corresponding to the primary antibodies into the circles to cover tissues after the sections are slightly dried, and incubating for 50min at room temperature in a dark place.
(20) Lining the cell nuclei: 1 mug/ml DAPI solution was stained in dark for about 3min and washed 3 times with PBS.
(21) Sealing piece: the tablets were sealed with glycerol.
(22) And (5) microscopic examination: and (5) performing fluorescence microscopy and image acquisition analysis.
As shown in a fourth graph, the method according to the invention is used for in vitro culture of glioma organoids, and glioma organoids derived from different patients show different morphological characteristics, but can be rapidly proliferated in vitro, and the glioma specific molecular marker is highly expressed.
A, glioma organoids three-dimensionally cultured examples;
B. C, the phase difference photo shows that the glioma organoids cultured by different individuals are different in morphology and compactness;
D, a glioma organoid HE staining result;
E. F, glioma organoid immunofluorescence staining results. (E) Ki67 positive indicates rapidly proliferating cells; (F) glioma-specific marker SOX2 positive cells.
Finally, it should be noted that the above-mentioned technical solution is only one embodiment of the present invention, and various modifications and variations can be easily made by those skilled in the art based on the application methods and principles disclosed in the present invention, and are not limited to the methods described in the above-mentioned specific embodiments of the present invention, therefore, the foregoing description is only preferred, and not meant to be limiting.
Claims (5)
1. A glioma organoid resuscitation fluid, said resuscitation fluid comprising 0.1-10 μg/ml taxifolin and 1-50 μg/ml calycosin.
2. A glioma organoid resuscitation medium comprising the glioma organoid resuscitation medium of claim 1.
3. The glioma organoid resuscitation broth of claim 2, wherein the method of preparing the glioma organoid resuscitation broth comprises the steps of:
(1) Preparing 1000 times concentrated glioma organoid resuscitation fluid mother liquor: accurately weighing taxifolin and calycosin in a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and gently shaking the centrifuge tube until the taxifolin and calycosin are dissolved, wherein mother liquor contains 0.1-10mg/ml taxifolin and 1-50mg/ml calycosin;
(2) And (3) filtering and sterilizing: the prepared mother solution is transferred into a laboratory through a material channel and is filtered and sterilized by a 0.22 mu m filter in a biosafety cabinet;
(3) Preparing a resuscitating culture solution: taking preheated glioma organoid growth culture solution, diluting the filtered sterilized recovery solution mother solution 1000 times according to the volume of the culture solution, and uniformly mixing for later use.
4. The glioma organoid resuscitation medium of claim 3, wherein said glioma organoid growth medium comprises DMEM high sugar cell medium, 1 xb 27 serum replacement, 20ng/mL EGF, 10mM niacinamide, 10 μΜ ascorbic acid, 25ng/mL FGF10, 5 μ M A83-01, 25ng/mL R-spondin-1.
5. A method for resuscitating and culturing a glioma-like device, characterized in that the method comprises resuscitating a frozen glioma-like organ by using the glioma-like organ resuscitating culture solution according to any one of claims 2 to 4, and comprises the following steps:
(1) Melting matrigel at 4deg.C in advance, placing on ice before experiment, pre-cooling gun head at-20deg.C in advance, and preheating culture dish in incubator in advance;
(2) Taking out the frozen glioma cells, immediately transferring the glioma cells into a water bath kettle at 37 ℃ for thawing, and shaking while thawing;
(3) Adding at least 5ml of precooled PBS into a centrifuge tube, adding the thawed cell suspension into the centrifuge tube, lightly mixing and centrifuging;
(4) Removing the supernatant, adding a proper amount of glioma organoid growth culture solution, and lightly suspending cells;
(5) Mixing the cell suspension and matrigel in a ratio of 1:5, dripping the mixture into a culture dish after uniformly mixing, taking care of not generating bubbles, standing for 3min, and putting the culture dish into an incubator for about 15min to solidify the matrigel;
(6) Taking out the culture dish, adding the prepared resuscitating culture solution into the culture dish, and placing the culture dish into an incubator for culturing;
(7) After 48 hours of culture, the glioma organoid growth medium is changed for continuous culture.
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