CN114480285A

CN114480285A - Recovery liquid, culture liquid and recovery culture method for glioma organoid

Info

Publication number: CN114480285A
Application number: CN202111442279.5A
Authority: CN
Inventors: 王拓; 高珂; 冒平
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-05-13
Anticipated expiration: 2041-11-30
Also published as: CN114480285B

Abstract

The invention provides a glioma organoid resuscitation solution, a culture solution and a resuscitation culture method, and belongs to the technical field of glioma organoid resuscitation. The resuscitation fluid comprises taxifolin and calycosin. The glioma organoid resuscitation liquid can greatly reduce the damage of DMSO to glioma organoids and the damage of DMSO to glioma organoids by freezing when the glioma organoids are resuscitated, can obviously improve the activity of glioma organoids, and improves the survival rate of glioma organoids after resuscitation; and does not contain animal serum and heterogenous source protein, and can avoid the pollution of virus, bacteria, mycoplasma and the like.

Description

Recovery liquid, culture liquid and recovery culture method for glioma organoid

Technical Field

The invention belongs to the technical field of glioma organoid resuscitation, and particularly relates to a glioma organoid resuscitation solution, a glioma organoid resuscitation culture solution and a glioma organoid resuscitation culture method.

Background

Glioma is a common malignant tumor of the central nervous system, mostly grows in an invasive growth mode, is not clearly demarcated with peripheral brain tissues, has high difficulty in complete excision after an operation and is easy to relapse after the operation, so the glioma is particularly important for in vitro research.

Organoid research is a recent in vitro culture technique for cells. The organoid model can well simulate the microenvironment of cells in vivo and has great advantages for the in vitro construction of research models with physiological functions. The in vitro culture of glioma organoids adopts a special 3D culture mode, but the cryopreservation method is a conventional cell cryopreservation method. The cell cryopreservation in a laboratory is 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, the toxicity can also poison and damage cells to a certain degree, the activity of the cells is reduced, and the damage to glioma organs is more obvious. Therefore, there is a need for an agent that can improve the viability and survival rate of glioma organoids during resuscitation of glioma organoids.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provide a glioma organoid resuscitation solution, and when the glioma organoid is resuscitated, the resuscitation solution is added into a culture solution, so that the activity and the survival rate of the glioma organoid can be improved, and the proliferation speed of the glioma organoid can be accelerated.

The second purpose of the invention is to provide a glioma organoid complex culture solution which can improve the activity and the survival rate of glioma organoids and accelerate the proliferation speed of glioma organoids.

The third purpose of the invention is to provide a recovery culture method of glioma organs.

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 principal ingredients of the glioma organoid resuscitation fluid comprise taxifolin with effective concentration range of 0.1-10 mug/ml and calycosin with effective concentration range of 1-50 mug/ml.

In a second aspect of the invention, a glioma organoid resuscitation culture solution is provided, which comprises the glioma organoid resuscitation culture solution.

Further, the preparation method of the glioma organoid resuscitation culture solution comprises the following steps:

(1) preparing 1000 times of concentrated glioma organoid resuscitation solution mother liquor: accurately weighing taxifolin and calycosin in a centrifugal tube by using an analytical balance, adding a proper amount of sterile water, and slightly shaking the centrifugal tube until the taxifolin and the calycosin are dissolved, wherein the mother liquor contains 0.1-10mg/ml of taxifolin and 1-50mg/ml of calycosin;

(2) and (3) filtering and sterilizing: transferring the prepared mother liquor into a laboratory through a material channel, and filtering and sterilizing in a biological safety cabinet by using a 0.22 mu m filter;

(3) preparing a recovery culture solution: taking preheated glioma organoid growth culture solution, diluting the filtered and sterilized resuscitation solution mother solution by 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, 1 XB 27 serum replacement, 20ng/mL EGF, 10mM nicotinamide, 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, there is provided a recovery culture method for a glioma organoid, wherein the recovery culture solution for a glioma organoid is used to recover a cryopreserved glioma organoid, the method comprising:

(1) melting matrigel at 4 ℃ in advance, placing on ice before experiment, precooling a gun head required by the experiment in a refrigerator at-20 ℃ in advance, and placing a required culture dish in an incubator for preheating in advance;

(2) taking out the frozen glioma cells, immediately transferring the glioma cells into a 37 ℃ water bath pan for thawing, and shaking the glioma cells while thawing;

(3) adding at least 5ml of precooled PBS into a centrifuge tube, adding the unfrozen cell suspension into the centrifuge tube, gently mixing uniformly and then centrifuging;

(4) removing the supernatant, adding a proper amount of glioma organoid growth culture solution, and gently suspending cells;

(5) mixing the cell suspension and the matrigel at a ratio of 1:5, dripping the mixture into a culture dish after uniformly mixing, taking care not to generate 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 resuscitation culture solution into the culture dish, and putting the resuscitation culture solution into an incubator for culture;

(7) after 48 hours of culture, the growth culture solution of the glioma organoid is changed for continuous culture.

Compared with the prior art, the invention has the beneficial effects that:

according to the glioma organoid resuscitation solution provided by the invention, when a glioma organoid is resuscitated, the damage of DMSO to the glioma organoid and the damage of DMSO to the glioma organoid caused by freezing can be greatly reduced, the activity of the glioma organoid can be remarkably improved, and the survival rate of the glioma organoid after resuscitation is improved.

The glioma organoid resuscitation solution provided by the invention does not contain animal serum and heterogenous source protein, and can avoid pollution of viruses, bacteria, mycoplasma and the like.

Drawings

FIG. 1 is an analysis of data from a CCK8 experiment optimized for taxifolin concentration;

FIG. 2 is an analysis of CCK8 experimental data for calycosin concentration optimization;

FIG. 3 is a data analysis of CCK8 experiments with different concentrations of drug combinations;

FIG. 4 shows immunohistochemical staining results.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings:

the invention provides a glioma organoid resuscitation liquid, which mainly comprises taxifolin and calycosin.

As a possible proposal, the principal ingredients of the glioma organoid resuscitation fluid comprise taxifolin with effective concentration range of 0.1-10 mug/ml and calycosin with effective concentration range of 1-50 mug/ml.

The cell culture method specifically uses culture solution added with resuscitation solution to culture cells, wherein the resuscitation solution contains taxifolin and calycosin, and the effective concentration range refers to the final use concentration of the taxifolin and calycosin in the culture solution.

Wherein, taxifolin is a flavanonol compound, belongs to vitamin P group, is a widely applied bioactive agent, and has various biological activities in human body like other flavonoid compounds, including antioxidation, free radical scavenging and other effects; especially, the existence of 4, 7-OH and 3 ', 4' -ortho-dihydroxy makes the antioxidant capacity of the biological flavonoid superior to that of the general biological flavonoid. Taxifolin was identified as one of the more potent vitamin P that could ensure survival of glioma organoids. The effective concentration range of the taxifolin is 0.1-10 mug/ml.

Calycosin has pharmacological effects of improving immunity, enhancing oxidation resistance, protecting glioma organoid, and promoting glioma organoid survival. The effective concentration range of calycosin of the invention is 1-50 mug/ml.

The invention also provides a glioma organoid resuscitation culture solution which comprises the glioma organoid resuscitation culture solution.

The preparation method of the glioma organoid resuscitation culture solution comprises the following steps:

(3) preparing a resuscitation culture solution, taking a preheated glioma organoid organ growth culture solution (the glioma organoid organ growth culture solution contains DMEM high-glucose cell culture solution, 1 XB 27 serum substitute, 20ng/mL EGF, 10mM nicotinamide, 10 mu M ascorbic acid, 25ng/mL FGF10, 5 mu M A83-01 and 25ng/mL R-spondin-1), diluting the resuscitation solution mother solution subjected to filtration sterilization by 1000 times according to the volume of the culture solution (which means diluting the resuscitation solution mother solution subjected to filtration sterilization by 1000 times by using the glioma organoid organ growth culture solution), and uniformly mixing for later use.

The invention also provides a recovery culture method of the glioma organoid, which is used for recovering the cryopreserved glioma organoid by using the recovery culture solution of the glioma organoid, and the method comprises the following steps:

(3) adding at least 5ml of precooled PBS into a centrifuge tube, adding the unfrozen cell suspension into the centrifuge tube, gently mixing uniformly, and centrifuging (1000rpm for 5 min);

(4) discarding the supernatant, adding appropriate amount of glioma organoid growth medium (the glioma organoid growth medium comprises DMEM high-glucose cell culture medium, 1 XB 27 serum substitute, 20ng/mL EGF, 10mM nicotinamide, 10 μ M ascorbic acid, 25ng/mL FGF10, 5 μ M A83-01, 25ng/mL R-spondin-1), and suspending the cells gently;

(6) taking out the culture dish, adding the prepared resuscitation culture solution into the culture dish, placing at 37 deg.C and 5% CO₂Culturing in an incubator;

The experimental methods in the following examples are conventional ones without specific explanation; the experimental reagents used were purchased from conventional reagent manufacturers without special instructions.

The experimental environment, experimental materials and instrument equipment which need to be prompted and explained in the invention are as follows:

1. the experimental environment is as follows: operating in a biological safety cabinet in a laboratory in a GMP environment.

2. Experimental reagent: calycosin, taxifolin, cell culture fluid and phosphate buffered saline PBS (PBS is the existing buffer solution and is not described in detail herein).

3. Instruments and equipment: CO 2₂Incubator, biological safety cabinet, enzyme-labeling instrument, centrifuge, electronic balance, 96-well plate.

Example one

A glioma organoid resuscitation liquid contains taxifolin with effective concentration range of 0.1-10 μ g/ml as main ingredient.

Specifically, in the present embodiment, the glioma organoid resuscitation fluid comprises taxifolin at a concentration of 0.1. mu.g/ml, 0.5. mu.g/ml, 1. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml.

Specifically, in this embodiment, the cells are glioma cells.

The method specifically comprises the following steps:

(1) melting matrigel at 4 deg.C in advance, placing on ice before experiment, pre-cooling the gun head required by experiment in a refrigerator at-20 deg.C in advance, and placing the required culture dish in an incubator in advance for preheating.

(2) Preparing a taxifolin mother solution: accurately weighing taxifolin into a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and slightly shaking the centrifuge tube until the taxifolin is dissolved. (the concentration of taxifolin mother liquor is 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 is filtered and sterilized in a biological safety cabinet by a filter with the diameter of 0.22 mu m.

(4) Preparing a pre-used resuscitation culture solution, taking a preheated 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) The frozen glioma cells are taken out and immediately transferred into a 37 ℃ water bath pan for thawing, and the shaking is carried out while the thawing is carried out.

(6) At least 5ml of precooled PBS was added to the centrifuge tube, and the thawed cell suspension was added to the centrifuge tube, gently mixed and centrifuged (1000rpm, 5 min).

(7) And (4) removing the supernatant, adding a proper amount of glioma organoid growth culture solution, and gently suspending the cells.

(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 not to generate bubbles, standing for 3min, and putting the culture dish into an incubator for about 15min to solidify the matrigel.

(9) Taking out the culture dish, adding a proper amount of the resuscitation culture solution prepared in the step 4 into the culture dish, placing the culture dish at 37 ℃ and 5% CO₂In an incubator.

(10) Culturing for 48 hr, and culturing with growth culture medium.

(11) Culturing the glioma organoid growth culture solution for 24 hours, then removing the culture solution, washing twice with PBS, adding matrigel lytic enzyme, placing on ice for digestion for 1-2 hours, transferring to a 15ml centrifuge tube, centrifuging at 1000rpm for 5min, and adding PBS for washing once.

(12) Centrifuging, removing supernatant, adding glioma organoid growth culture solution to suspend cells, and transferring to a 96-well plate with 100 μ l cell suspension per well.

(13) Each well was supplemented with 10. mu.l of CCK8 reagent, and the mixture was incubated at 37 ℃ for 2 hours.

(14) And detecting the light absorption value at 450nm by using a microplate reader.

Example two

A glioma organoid resuscitation liquid contains calycosin with effective concentration range of 1-50 μ g/ml as main ingredient.

Specifically, in this example, the glioma organoid resuscitation fluid comprises calycosin at concentrations of 1. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 50. mu.g/ml.

Specifically, in this embodiment, the cells are glioma cells.

The method specifically comprises the following steps:

(2) Preparing a calycosin mother solution: accurately weighing calycosin into a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and slightly shaking the centrifuge tube until the calycosin is dissolved. (the concentration of calycosin mother liquor is 1mg/ml, 5mg/ml, 10mg/ml, 20mg/ml, 50mg/ml)

(3) And (3) filtering and sterilizing: transferring the prepared calycosin mother liquor into a laboratory through a material channel, and filtering and sterilizing in a biological safety cabinet by using a 0.22 mu m filter.

(4) Preparing a pre-used resuscitation culture solution, taking a preheated 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.

(6) Adding at least 5ml of precooled PBS or glioma organoid growth culture medium into a centrifuge tube, adding the thawed cell suspension into the centrifuge tube, gently mixing uniformly, and centrifuging (1000rpm for 5 min).

(8) Mixing the cell suspension and matrigel at a ratio of 1:5, mixing, dripping the mixture into a culture dish, keeping away bubbles, standing for 3min, and placing the culture dish into an incubator for about 15min to solidify the matrigel.

(9) Taking out the culture dish, adding a proper amount of the culture solution prepared in the step 4 into the culture dish, placing the culture dish at the temperature of 37 ℃ and adding 5% CO₂In an incubator.

(10) Culturing for 48 hr, and culturing with growth culture medium.

EXAMPLE III

A glioma organoid resuscitation liquid contains taxifolin with effective concentration range of 0.1-10mg/ml and calycosin with effective concentration range of 1-50mg/ml as main ingredients.

Specifically, in this example, the glioma organoid resuscitation fluid comprises 5. mu.g/ml taxifolin and 1. mu.g/ml calycosin, 5. mu.g/ml taxifolin and 10. mu.g/ml calycosin, 5. mu.g/ml taxifolin and 50. mu.g/ml calycosin, 0.1. mu.g/ml taxifolin and 10. mu.g/ml calycosin, 1. mu.g/ml taxifolin and 10. mu.g/ml calycosin, 10. mu.g/ml taxifolin and 10. mu.g/ml calycosin.

Specifically, in this embodiment, the cells are glioma cells.

The method specifically comprises the following steps:

(2) Preparing a glioma organoid resuscitation solution mother solution: accurately weighing taxifolin and calycosin in a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and slightly shaking the centrifuge tube until the taxifolin and the calycosin are dissolved. (concentrations of taxifolin and calycosin in mother liquor of glioma organoid resuscitation solution are 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, respectively)

(3) And (3) filtering and sterilizing: the prepared mother liquor of the glioma organoid resuscitation solution is transferred into a laboratory through a material channel, and is filtered and sterilized by a filter with the diameter of 0.22 mu m in a biological safety cabinet.

(4) Preparing a pre-used resuscitation culture solution, taking a preheated glioma organoid growth culture solution, diluting the glioma organoid resuscitation solution mother solution by 1000 times according to the volume of the culture solution, and uniformly mixing for later use.

(10) Culturing for 48 hr, and culturing with growth culture medium.

(13) Mu.l of CCK8 reagent was added to each well, and the mixture was incubated at 37 ℃ for 2 hours in an incubator.

Comparative example

A glioma organoid resuscitation liquid contains glioma organoid growth culture solution, and does not contain taxifolin and calycosin.

Specifically, in this embodiment, the cells are glioma cells.

The method specifically comprises the following steps:

(2) The frozen glioma cells are taken out and immediately transferred into a 37 ℃ water bath pan for thawing, and the pan is shaken while thawing.

(3) And (3) adding at least 5ml of precooled PBS or glioma organoid growth culture solution into a centrifuge tube, adding the unfrozen cell suspension into the centrifuge tube, gently mixing uniformly, and centrifuging (1000rpm for 5 min).

(4) And (4) removing the supernatant, adding a proper amount of glioma organoid growth culture solution, and gently suspending the cells.

(5) Mixing the cell suspension and matrigel at a ratio of 1:5, mixing, dripping the mixture into a culture dish, keeping away bubbles, standing for 3min, and placing the culture dish into an incubator for about 15min to solidify the matrigel.

(6) Taking out the culture dish, adding appropriate amount of culture solution for glioma organoid growth, placing at 37 deg.C and 5% CO₂In an incubator.

(7) Culturing for 48 hr, and culturing with growth culture medium.

(8) After further culturing for 24 hours, the culture solution is discarded, washed twice with PBS, matrigel-dissolving enzyme is added, the mixture is placed on ice for digestion for 1 to 2 hours, then the mixture is transferred to a 15ml centrifuge tube, centrifuged for 5min at 1000rpm, and washed once with PBS.

(9) Centrifuging, removing supernatant, adding glioma organoid growth culture solution to suspend cells, and transferring to a 96-well plate with 100 μ l cell suspension per well.

(10) Each well was supplemented with 10. mu.l of CCK8 reagent, and the mixture was incubated at 37 ℃ for 2 hours.

(11) And detecting the light absorption value at 450nm by using a microplate reader.

Example one comparison with the control example shows the results in fig. 1, and table 1 shows the experimental data of CCK8 optimized for taxifolin concentration.

TABLE 1

As shown in fig. 1 and table 1, when the glioma cells are recovered, the paclitaxel with different concentrations is added into the culture solution, the cell proliferation activity is stronger with the increase of the concentration in the range of 0 μ g/ml to 5 μ g/ml, the cell proliferation activity is strongest when the concentration is 5 μ g/ml, and the cell proliferation activity is in a flat trend in the range of 5 μ g/ml to 10 μ g/ml, so that 5 μ g/ml of paclitaxel is selected as the optimal concentration.

The results of comparing example two with the control example are shown in fig. 2, and table 2 shows the experimental data of CCK8 optimized for taxifolin concentration.

As shown in FIG. 2 and Table 2, when the glioma cells are recovered, the calycosin with different concentrations is added into the culture solution, the cell proliferation activity is stronger along with the increase of the concentration within the range of 0 mu g/ml to 10 mu g/ml, the cell proliferation activity is strongest when the concentration is 10 mu g/ml, and the cell proliferation activity is reduced within the range of 10 mu g/ml to 50 mu g/ml, so that the calycosin with the concentration of 10 mu g/ml is selected as the optimal concentration.

TABLE 2

The results of comparing example three with the control example are shown in fig. 3, and table 3 shows the experimental data of CCK8 optimized for taxifolin concentration.

TABLE 3

As shown in fig. 3 and table 3, when the glioma cells are resuscitated, taxifolin and calycosin with different concentrations are added into the culture solution, and the results show that the combination with different concentrations can improve the cell proliferation activity to different degrees, wherein the combination of 5 μ g/ml taxifolin and 10 μ g/ml calycosin has a particularly significant improvement on the cell proliferation activity, and therefore, the cell activity enhancing solution of the present invention adopts 5 μ g/ml taxifolin and 10 μ g/ml calycosin as the optimal conditions.

Blank example

A glioma organoid resuscitation liquid contains taxifolin with effective concentration range of 0.1-10 μ g/ml and calycosin with effective concentration range of 1-50 μ g/ml.

Specifically, in this example, the glioma organoid resuscitation fluid does not contain taxifolin and does not contain calycosin.

(1) Adding cell culture fluid into 96-well plate at 100 μ l per well, placing at 37 deg.C and 5% CO₂In an incubator.

(2) After 48 hours of culture, the growth culture solution of the glioma organoid is changed for continuous culture.

(3) After 24 hours, the culture medium was discarded, and 100. mu.l of the culture medium was added to each well, followed by 10. mu.l of CCK8 reagent, and the mixture was incubated at 37 ℃ for 2 hours in an incubator.

(4) And detecting the light absorption value at 450nm by using a microplate reader.

Table 4 shows the experimental data of CCK8 for the blank group.

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 example are mainly used for calculating the absorbance of the first example, the second example, the third example and the control example, and the background absorbance interference of the glioma organoid growth culture solution in the blank holes is eliminated and subtracted during calculation.

Example four morphological and immunofluorescence identification of glioma Primary cells

Specifically, in the present example, the principal components of the glioma organoid resuscitation fluid comprise taxifolin with an effective concentration of 5 μ g/ml and calycosin with an effective concentration of 10 μ g/ml.

Specifically, in this example, the cells are primary glioma cells.

Specifically, in this example, the primary antibody is a rabbit anti-human (SOX2) 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) the matrigel is put into a refrigerator at the temperature of minus 20 ℃ in advance to be melted in advance, the gun is placed on ice before the experiment, and a required culture dish is put into an incubator in advance to be preheated.

(2) Preparing a glioma organoid resuscitation solution mother solution: accurately weighing taxifolin and calycosin in a centrifuge tube by using an analytical balance, adding a proper amount of sterile water, and slightly shaking the centrifuge tube until the taxifolin and the calycosin are dissolved. (the concentration of taxol and calycosin in the mother liquor of the glioma organoid resuscitation liquid is 5mg/ml and 10mg/ml)

(10) Culturing for 48 hr, and culturing with growth culture medium.

(11) Culturing for three weeks until the glioma organoid grows to 2 mm.

(12) Paraffin embedded sections were prepared.

(13) Baking slices: and taking paraffin embedded sections, and baking the sections in an oven for 2 hours.

(14) Paraffin embedded sections were deparaffinized and hydrated: placing the slices in xylene I15 min-xylene II 15 min-xylene III 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% ethyl alcohol 5 min-75% ethyl alcohol 5 min-distilled water washing.

(15) Antigen retrieval: placing the tissue slices in a repairing box filled with citric acid antigen repairing buffer solution (pH6.0) in a microwave oven for antigen repairing, stopping heating for 8min until boiling, maintaining the temperature, and turning to low and medium heat for 7min to prevent excessive evaporation of the buffer solution. After natural cooling, the slide was washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.

(16) Blocking endogenous peroxidase: the sections were placed in 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH7.4) with shaking on a destaining shaker.

(17) And (5) serum blocking, namely dripping 3% BSA (bovine serum albumin) into a histochemical ring to uniformly cover the tissues, and blocking for 30min at room temperature.

(18) Adding a primary antibody: gently removing the confining liquid, dripping PBS (phosphate buffer solution) on the slices to prepare primary antibodies according to a certain proportion, and flatly placing the slices in a wet box for incubation at 4 ℃ overnight. (Small amount of water added in wet box to prevent evaporation of antibody)

(19) Adding a secondary antibody: slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. After the section is slightly spin-dried, a secondary antibody (marked by Alexa 667) which is corresponding to the primary antibody is dripped into the ring to cover the tissue, and the tissue is incubated for 50min at room temperature in the dark.

(20) Lining and staining cell nucleus: the DAPI solution of 1 ug/ml was stained in the dark for about 3min and washed 3 times with PBS.

(21) Sealing: the sheets were mounted with glycerol.

(22) Microscopic examination: and (4) performing microscopic examination by using a fluorescence microscope, and collecting and analyzing images.

As shown in the figure, when glioma organoids are cultured in vitro according to the method of the invention, the glioma organoids from different patients show different morphological characteristics, but can rapidly proliferate in vitro and highly express glioma-specific molecule marker.

A, a glioma organoid three-dimensional culture example;

B. c, phase difference pictures show that glioma organoids cultured by different individuals have different shapes and different compactness degrees;

d, staining results of glioma organoid HE;

E. f, immunofluorescence staining results of glioma organoids. (E) Ki67 positive indicates rapidly proliferating cells; (F) glioma-specific marker SOX2 positive cells were shown.

Finally, it should be noted that the above-mentioned technical solution is only one embodiment of the present invention, and it will be apparent to those skilled in the art that various modifications and variations can be easily made based on the application method and principle of the present invention disclosed, and the method is not limited to the above-mentioned specific embodiment of the present invention, so that the above-mentioned embodiment is only preferred, and not restrictive.

Claims

1. A glioma organoid resuscitation fluid comprising taxifolin and calycosin.

2. The glioma organoid resuscitation fluid of claim 1 wherein said glioma organoid resuscitation fluid comprises, as major components, taxifolin at an effective concentration ranging from 0.1 to 10 μ g/ml and calycosin at an effective concentration ranging from 1 to 50 μ g/ml.

3. A glioma organoid resuscitation culture fluid, which comprises the glioma organoid resuscitation culture fluid.

4. The glioma organoid resuscitation culture fluid of claim 3, wherein the preparation method of the glioma organoid resuscitation culture fluid comprises the following steps:

5. The glioma organoid resuscitation culture fluid of claim 4, wherein said glioma organoid growth culture fluid comprises DMEM high glucose cell culture fluid, 1 XB 27 serum replacement, 20ng/mL EGF, 10mM nicotinamide, 10 μ M ascorbic acid, 25ng/mL FGF10, 5 μ M A83-01, 25ng/mL R-spondin-1.

6. A method for resuscitating a glioma organoid comprising resuscitating a cryopreserved glioma organoid with the glioma organoid resuscitating culture medium of any of claims 3-5, comprising:

(3) adding at least 5ml of precooled PBS into a centrifuge tube, adding the thawed cell suspension into the centrifuge tube, gently mixing uniformly and centrifuging;