A co-culture model of brain glioma organoids and mouse brain slices and application thereof

Abstract

A co-culture model of brain glioma organoids and mouse brain slices, characterized in that the co-culture model is prepared by the following method: (1) taking mouse brain slices, culturing to obtain cultured mouse brain slices; (2) injecting brain glioma organoids into the cultured mouse brain slices obtained in step (1) to obtain brain slices injected with brain glioma organoids; (3) culturing the brain slices injected with brain glioma organoids obtained in step (2) to obtain the co-culture model. The co-culture model of the application can be used to evaluate the invasion phenotype of glioma organoids in vitro and to test and screen brain glioma drugs.

Description

Invention Field

[0001] This application belongs to the field of cell biology. Specifically, this application provides a co-culture model of glioma organoids and mouse brain slices and its application. Background Technology

[0002] Tumor organoids are three-dimensional in vitro models of tumors cultivated using organoid technology, possessing tissue structure and function similar to actual tumors. They retain the intratumoral heterogeneity and surrounding microenvironment to the greatest extent possible, allowing for long-term stable passage and self-renewal and self-assembly during passage and proliferation, thus performing the corresponding physiological functions of a tumor. Their culture cycle is short, success rate is high, and they require very little tissue, making them particularly suitable for gliomas with high intratumoral heterogeneity.

[0003] Gliomas are the most common malignant tumors of the central nervous system, with poor prognosis, severe problems with tolerance to radiotherapy and chemotherapy, and a lack of corresponding in vitro models to characterize their malignant biological features. The establishment of glioma organoids can assist in in vitro "surrogate drug testing"; however, unlike traditional cell line models, the invasive phenotype of organoids is difficult to verify visually in vitro.

[0004] Brain slices are axial sections of the incompletely developed brain tissue of newborn mice, which are then continuously cultured in vitro in chambers. Traditional slicing methods require cryostats, are cumbersome, and cannot rapidly transfer and culture the tissue. Furthermore, traditional culture formulations are primarily used for the culture of the telencephalon, and are rarely used for the preparation and culture of mouse hindbrain slices. Traditional brain slice culture is mainly used in the field of neurobiology research, and further exploration and expansion of its research scope are urgently needed.

[0005] Co-culturing glioma organoids with mouse brain slices allows for convenient in vitro characterization of the invasiveness of different types of gliomas, simulating and studying their malignant biological characteristics. Simultaneously, this co-culture model enables dual screening of drug safety and efficacy, greatly improving screening efficiency. Summary of the Invention

[0006] On the one hand, this application provides a co-culture model of glioma organoids and mouse brain slices, the co-culture model being prepared using the following method:

[0007] (1) Take mouse brain slices, culture them, and obtain cultured mouse brain slices;

[0008] (2) Inject the glioma organoids into the cultured mouse brain slices obtained in step (1) to obtain brain slices injected with glioma organoids;

[0009] (3) Brain slices obtained from step (2) that were injected into glioma organoids.

[0010] The co-culture model described in this application refers to the culture product obtained through cultivation.

[0011] Furthermore, the mice mentioned in step (1) are newborn ICR strain mice that are 1-2 weeks old.

[0012] Furthermore, the brain slices mentioned in step (1) are telencephalon or brainstem slices.

[0013] Further, in step (1), mouse brain slices are taken and transferred to MF microporous membranes and cultured for more than one week.

[0014] Further, in step (1), a DMEM high-glucose medium containing 20 ng / mL EGF, 20 ng / mL bFGF, 100 U / mL penicillin, 100 μg / mL streptomycin, 1×N2 additive, and 1×B27 additive was used.

[0015] Furthermore, the glioma organoids are WHO grade I-IV glioma organoids derived from patient tissue.

[0016] Further, in step (2), after using a pipette to aspirate the glioma organoids, it is inserted obliquely at 45° into the mouse brain slice, slowly injected, and withdrawn after ensuring that the glioma organoids do not escape.

[0017] Further, in step (3), the medium containing 2 mmol / L L-glutamine substitute, 1×N2 additive, 1×B27 additive, 500 μmol / L β-mercaptoethanol, 100 μmol / L non-essential amino acids, 100×ITS 1X, 100U / mL penicillin, 100μg / mL streptomycin, 20 ng / mL EGF, and 20 ng / mL bFGF was first cultured for 48 hours using an equal mixture of Neurobasal medium and DMEM / F12 medium; then, the medium was cultured for 1-2 weeks using DMEM high-glucose medium containing 20 ng / mL EGF, 20 ng / mL bFGF, 100U / mL penicillin, 100μg / mL streptomycin, 1×N2 additive, and 1×B27 additive.

[0018] On the other hand, this application provides a method for preparing the above-mentioned glioma organoid co-culture model with mouse brain slices.

[0019] On the other hand, this application provides the application of the above co-culture model in evaluating the in vitro invasive phenotype of glioma organoids.

[0020] The so-called invasive phenotype refers to the ability of glioma tumors to invade normal tissues and cause local infiltration and migration. The evaluation method is to perform pathological staining on the co-culture model to characterize human tumor cells, and calculate the degree of migration of tumor cells away from the tumor center to measure the invasiveness of glioma organoids.

[0021] Specifically, the co-cultured model was fixed overnight with 4% paraformaldehyde, then dehydrated, embedded, and sectioned, and subjected to HE staining and STEM121 human marker staining.

[0022] In each of at least three slices (or three co-culture model operations), human tumor cells labeled in at least five directions are selected. Using ImageJ software, the main body of the glioma organoid is fitted with a circle, and the center and outline of the circle are marked. The distance L from each selected human tumor cell to the center of the marked circle is measured using ImageJ software, and the radius R of the marked circle is measured. The calculation formula is as follows:

[0023] Invasion level = AVG (average) [(LR) / R] × 100%.

[0024] On the other hand, this application provides the application of the above-mentioned co-culture model in in vitro drug testing or screening for gliomas.

[0025] The test or screening involves testing drug sensitivity on a co-culture model while simultaneously observing the drug's sensitivity to tumor cells and normal cells.

[0026] Specifically, the target drug was applied to the culture medium and could be administered repeatedly. After the drug intervention was completed, the cells were fixed overnight in 4% paraformaldehyde, dehydrated, embedded, sectioned, and stained with hematoxylin and eosin (HE) or immunofluorescence to observe the drug killing effect on tumor and normal mouse brain cells, and to detect drug sensitivity and safety.

[0027] On the other hand, this application provides a mouse brain slice culture medium, wherein the culture medium is a DMEM high glucose medium comprising 20 ng / mL EGF, 20 ng / mL bFGF, 100 U / mL penicillin, 100 μg / mL streptomycin, 1×N2 additive, and 1×B27 additive.

[0028] On the other hand, this application provides a buffered culture medium, which is a medium comprising equal volumes of L-glutamine substitute 2 mmol / L, 1×N2 additive, 1×B27 additive, β-mercaptoethanol 500 μmol / L, non-essential amino acids 100 μmol / L, 100×ITS 1X, 100U / mL penicillin, 100μg / mL streptomycin, EGF 20 ng / mL, bFGF 20 ng / mL, Neurobasal medium and DMEM / F12 medium.

[0029] This invention simplifies and optimizes mouse brain slice culture methods, using specialized culture media and mouse brain cutting molds to efficiently prepare sustainably cultured mouse brain slices in vitro, and establishes a co-culture model of glioma organoids and mouse brain slices. This model can characterize the invasiveness of glioma organoids and assess their invasive phenotype by calculating the degree of tumor cell migration. In drug testing, it can be used to evaluate the sensitivity of drugs to glioma organoids and normal brain tissue, exhibiting high efficiency and accuracy. Therefore, this invention provides a novel experimental platform and method for glioma invasiveness research and preclinical drug testing. Attached Figure Description

[0030] Figure 1 This is the result of the simplified co-culture model of mouse brain slices, glioma organoids and mouse brain slices of the present invention.

[0031] Figure 2 This is the pathological staining result of mouse brain slices cultured in this invention.

[0032] Figure 3 These are the pathological staining results of the co-culture model of glioma organoids and mouse brain slices established in this invention.

[0033] Figure 4 Examples and results of the in vitro invasive phenotype evaluation method for glioma organoids provided by this invention;

[0034] Figure 5 The results are from the in vitro preclinical drug testing method for gliomas based on the above co-culture model provided by this invention. Detailed Implementation

[0035] Example 1: Mouse brain slice culture method:

[0036] (1) A special culture medium for mouse brain slices was prepared according to the present invention. The culture medium components include: 500 mL of DMEM high glucose medium (Gibco, 11995073), 20 ng / mL EGF (Cyento SC102), 20 ng / mL bFGF (Cyento SC107-10 μg), 100 U / mL penicillin and 100 μg / mL streptomycin (100×, Thermo Fisher Scientific 10378016), 1× N2 additive (50×, Cyento S60314017A), and 1× B27 additive (50×, Cyento S60314015A).

[0037] (2) A simplified method for in vitro culture of mouse brain slices according to the present invention:

[0038] I. Feed the female rats with tethers (Sberger, ICR strain) and observe them daily until 7 days after the newborn mice are born (which can be extended to 14 days). Then, place 6 newborn mice on ice for anesthesia according to their birth age (7 days) or administer 50-150 mg / kg ketamine via intraperitoneal injection for anesthesia (8-14 days).

[0039] II. After satisfactory anesthesia, the mouse head was cut off with a large tissue cutter, quickly rinsed in pre-cooled 75% medical alcohol (Anjie Gaoke), and then thoroughly rinsed in pre-cooled artificial cerebrospinal fluid (Scientoo, SSL6630) to remove the alcohol.

[0040] III. Use sterile forceps and ophthalmic scissors to separate soft tissues and thin, soft skull bones, expose the telencephalon and brainstem, separate and sever the olfactory nerve connection to the telencephalon, separate and sever the cranial nerves originating from the brainstem, obtain complete mouse brain tissue, and temporarily store it in pre-cooled artificial cerebrospinal fluid.

[0041] IV. Prepare the blade in advance by disinfecting it in pre-cooled 75% medical alcohol and then temporarily storing it in pre-cooled artificial cerebrospinal fluid;

[0042] V. Place the dissected mouse brain in a mouse brain cutting mold that has been pre-sterilized by autoclaving, with the ventral side facing down; insert two blades into the gap between adjacent brain cutting molds and cut; remove the cephalic blade, which carries away brain tissue from the cephalic region as unwanted waste; remove the caudal blade, and attach the target brain slice to the cephalic side of the caudal blade.

[0043] VI. Cut three pieces consecutively. After removing the first blade, immediately place it in the next cutting die slot on the tail side of the last blade. Repeat the above steps to remove pieces.

[0044] VII. After taking the slides, rinse the brain slides with pre-cooled artificial cerebrospinal fluid into a large dish containing pre-cooled artificial cerebrospinal fluid. After all slides have been taken, carefully transfer the brain slides onto MF microporous membranes (Millipore, PICM03050) using sterile blunt forceps, with 3 brain slides per membrane, and then place them in a six-well plate.

[0045] VIII. Add 1.2-1.5 mL of the special culture medium to each well and incubate continuously in an incubator at 37℃ and 5% CO2.

[0046] IX. After 24 hours, examine the morphology of the brain slices, remove those with poor growth, change the medium, and continue culturing; then change the medium every 3 days, and perform subsequent experiments after 1 week of in vitro culture. Figure 1 ).

[0047] X. Cultured mouse brain slices were stained with hematoxylin and eosin (HE) and subjected to multiplex immunofluorescence staining. Figure 2 ).

[0048] Example 2: Co-culture model of glioma organoids and mouse brain slices.

[0049] I. Culture mouse brain slices in vitro using the method described in Example 1;

[0050] II. The cultured glioma organoids (derived from a patient with pilocytic astrocytoma, CNS WHOI grade, approximately 200 μm in diameter) were aspirated using the smallest range pipette tip (with part of the tip cut off), and carefully inserted at an angle of approximately 45° into the mouse brain slice. The injection was slow and the organoids were withdrawn after confirming that there was no leakage.

[0051] III. Culture using buffered medium prepared according to the following components: Neurobasal Medium 250 mL (Gibco, 21103049), DMEM / F12 medium 250 mL (Cyintu, C11330500BT), L-glutamine substitute 2 mmol / L (Cyintu, S60314014A), 1×N2 additive (50×, Cyintu S60314017A), 1×B27 additive (50×, Cyintu S60314015A), β-mercaptoethanol 500 μmol / L (Cyintu, S60314019A), non-essential amino acids 100 μmol / L (Cyintu, S60314011A), 100×ITS 1X (Cyenta, SC25800), 100 U / mL penicillin and 100 μg / mL streptomycin (100×, Thermo Fisher Scientific 10378016), EGF 20 ng / mL (Cyenta SC102), bFGF 20 ng / mL (Cyenta SC107-10 μg).

[0052] IV. After 48 hours, replace with brain slice-specific culture medium and incubate at 37°C with 5% CO2. Change the medium every 3 days thereafter. Perform subsequent invasive phenotype assessment or drug testing experiments after 1-2 weeks. Figure 1 ).

[0053] Examples 3-4: Co-culture models of glioma organoids and mouse brain slices

[0054] The methods in Examples 3 and 4 are the same as in Example 2, except that the glioma organoids in Example 3 were derived from a patient with astrocytoma and IDH mutation (CNS WHO grade II), and the glioma organoids in Example 3 were derived from a patient with diffuse midline glioma and H3K27M mutation (CNS WHO grade IV).

[0055] HE and multiplex immunofluorescence staining were performed on the organoid brain slice co-culture models cultured in Examples 2-4, as well as the source organoids and parental tissues. The co-culture models preserved the complexity of the tumorigenic microenvironment and more realistically simulated the growth characteristics of tumors in vivo. Figure 3 ).

[0056] Example 5: In vitro invasiveness phenotype evaluation method for glioma organoids

[0057] I. The organoid and mouse brain slice co-culture model established using Examples 2 to 4 of this invention;

[0058] II. After 1 week of culture, fix, dehydrate and embed, prepare paraffin slides, stain with HE, and calculate the degree of migration of tumor cells away from the tumor center in five directions on each of at least three slides (or three co-culture models) to measure the invasiveness of glioma organoids.

[0059] III. Using ImageJ software, fit the main body of the glioma organoid with a circle, mark the center and outline of the circle, and use ImageJ software to measure the distance L from each selected human tumor cell to the center of the marked circle in each direction. Measure the radius R of the marked circle and calculate the following formula: Invasiveness = AVG (average) [(LR) / R] × 100%.

[0060] IV. The invasive phenotype of glioma organoids as measured by the organoid-mouse brain slice co-culture model established in Examples 2-4 is as follows: Figure 4 As shown, the most malignant type, diffuse midline glioma (the glioma organoid in Example 4 was derived from a case of diffuse midline glioma with H3K27M mutation), exhibited the highest degree of invasion. Figure 4 ).

[0061] Example 6: Co-culture model for in vitro preclinical drug testing of glioma

[0062] I. A co-culture model of organoids and mouse brain slices established using this invention;

[0063] II. Add the target concentration of the drug (e.g., 10 nM Panobinostat) to the brain slice-specific culture medium and incubate continuously in a 37℃ 5% CO2 incubator.

[0064] III. After one week of continuous culture, the tumor was fixed, dehydrated, embedded, and prepared into paraffin sections. HE staining was performed to observe the growth of tumor organoids and the morphology of mouse brain cells, in order to verify the efficacy and safety of the drug. Figure 5 As shown, Panobinostat is a potentially effective drug, causing the tumor at the organoid inoculation site (center) to disappear and form a cavity.

Claims

1. A co-culture model of glioma organoids and mouse brain slices, characterized in that, The co-culture model was prepared using the following method: (1) Mouse brain slices were obtained and transferred to MF microporous membranes; cultured in DMEM high-glucose medium containing 20 ng / mL LEGF, 20 ng / mL bFGF, 100 U / mL penicillin, 100 μg / mL streptomycin, 1×N2 additive and 1×B27 additive for more than 1 week to obtain cultured mouse brain slices; the mice were ICR strain mice 1-2 weeks old; the brain slices were telencephalon or brainstem brain slices; (2) After aspirating the glioma organoids with a pipette, insert it at a 45° angle into the cultured mouse brain slice obtained in step (1). Slowly inject the organoids to ensure that they do not escape before withdrawing the pipette to obtain a brain slice containing the injected glioma organoids. The glioma organoids are WHO grade I-IV pathological glioma organoids derived from the patient's tissue. (3) The brain slices injected with glioma organoids obtained in step (2) were cultured for 48 hours in a medium containing 2 mmol / L L-glutamine substitute, 1×N2 additive, 1×B27 additive, 500 μmol / L β-mercaptoethanol, 100 μmol / L non-essential amino acids, 1X ITS, 100 U / mL penicillin, 100 μg / mL streptomycin, 20 ng / mL EGF and 20 ng / mL bFGF, and an equal mixture of Neurobasal medium and DMEM / F12 medium. Subsequently, the brain slices were cultured in DMEM high-glucose medium containing 20 ng / mLEGF, 20 ng / mL bFGF, 100 U / mL penicillin, 100 μg / mL streptomycin, 1×N2 additive and 1×B27 additive for 1-2 weeks to obtain the co-culture model.

2. A method for preparing a co-culture model of glioma organoids and mouse brain slices, characterized in that, The method includes: (1) Mouse brain slices were obtained and transferred to MF microporous membranes; cultured in DMEM high-glucose medium containing 20 ng / mL LEGF, 20 ng / mL bFGF, 100 U / mL penicillin, 100 μg / mL streptomycin, 1×N2 additive and 1×B27 additive for more than 1 week to obtain cultured mouse brain slices; the mice were ICR strain mice 1-2 weeks old; the brain slices were telencephalon or brainstem brain slices; (2) After aspirating the glioma organoids with a pipette, insert it at a 45° angle into the cultured mouse brain slice obtained in step (1). Slowly inject the organoids to ensure that they do not escape before withdrawing the pipette to obtain a brain slice containing the injected glioma organoids. The glioma organoids are WHO grade I-IV pathological glioma organoids derived from the patient's tissue. (3) The brain slices injected with glioma organoids obtained in step (2) were cultured for 48 hours in a medium containing 2 mmol / L L-glutamine substitute, 1×N2 additive, 1×B27 additive, 500 μmol / L β-mercaptoethanol, 100 μmol / L non-essential amino acids, 1×ITS, 100 U / mL penicillin, 100 μg / mL streptomycin, 20 ng / mL EGF and 20 ng / mL bFGF, and equal volumes of Neurobasal medium and DMEM / F12 medium. Subsequently, the brain slices were cultured in DMEM high-glucose medium containing 20 ng / mLEGF, 20 ng / mL bFGF, 100 U / mL penicillin, 100 μg / mL streptomycin, 1×N2 additive and 1×B27 additive for 1-2 weeks to obtain the co-culture model.

3. The application of the co-culture model according to claim 1 in evaluating the in vitro invasive phenotype of glioma organoids.

4. The application according to claim 3, wherein the co-culture model is fixed overnight with 4% paraformaldehyde, dehydrated, embedded, and sectioned, and HE staining and STEM121 human marker staining are performed; in each of at least three sections, human tumor cells labeled in at least five directions are selected, and the main body of the glioma organoid is fitted with a circle using ImageJ software, marking the center and outline of the circle; the distance L from each selected human tumor cell to the center of the marked circle is measured using ImageJ software, and the radius R of the marked circle is measured; the invasiveness is calculated using the formula: invasiveness = [(LR) / R] × 100%.

5. The application of the co-culture model according to claim 1 in in vitro drug testing or screening for gliomas.

6. The application according to claim 5, wherein the target drug is applied to the culture medium of the co-culture model; after the drug intervention is completed, the cells are fixed overnight with 4% paraformaldehyde, dehydrated, embedded, sectioned, and subjected to HE staining or immunofluorescence staining; the drug killing effect on tumor and normal mouse brain cells is observed, and the drug sensitivity and safety are detected.

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