Brain glioma organoid and mouse brain slice co-culture model and application

Abstract

The invention relates to a brain glioma organoid and mouse brain slice co-culture model, which is characterized in that the co-culture model is prepared by the following method: (1) taking a mouse brain slice, and culturing to obtain a cultured mouse brain slice; (2) injecting a brain glioma organ into the cultured mouse brain slice obtained in the step (1) to obtain a brain slice into which the brain glioma organ is injected; and (3) culturing the brain slice injected into the brain glioma organ obtained in the step (2) to obtain the co-culture model. The co-culture model can be used for in-vitro evaluation of glioma organ invasion phenotypes and brain glioma drug testing and screening.

Description

Field of the Invention

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

[0002] Tumor organoids are three-dimensional in vitro tumor models cultivated using organoid technology, with tissue structure and function similar to actual tumors. They maximize the preservation of the internal heterogeneity of the tumor and the surrounding microenvironment, allowing for long-term stable passage and the ability to self-renew and self-assemble during proliferation, fulfilling the physiological functions of the tumor. They have a short culture cycle, high success rate, and minimal tissue requirement, making them particularly suitable for gliomas, which exhibit high intratumoral heterogeneity.

[0003] Glioma is the most common malignant tumor of the central nervous system, with a poor prognosis and serious challenges with radiotherapy and chemotherapy tolerance. Furthermore, there is a lack of in vitro models to characterize its malignant biological characteristics. The establishment of glioma organoids can facilitate in vitro "surrogate drug testing," but unlike traditional cell line models, their invasive phenotype is difficult to directly verify in vitro.

[0004] Brain slices are prepared by axially sectioning the immature brain tissue of newborn mice and continuously culturing them in a chamber outside the body. Traditional slicing methods require the use of a freezing microtome, which is cumbersome and cannot be quickly transferred to culture. Furthermore, traditional culture recipes are primarily used for culturing the telencephalon and are rarely used for culturing mouse hindbrain slices. Traditional brain slice culture is primarily used in neurobiology research and urgently needs further exploration and expansion.

[0005] Co-culturing brain glioma organoids with mouse brain slices can conveniently characterize the aggressiveness of different types of gliomas in vitro, simulate and study their malignant biological characteristics; at the same time, this co-culture model can simultaneously achieve dual screening of drug safety and efficacy, greatly improving screening efficiency. Summary of the Invention

[0006] In one aspect, the present application provides a co-culture model of glioma organoids and mouse brain slices, wherein the co-culture model is prepared using the following method:

[0007] (1) obtaining mouse brain slices, culturing them, and obtaining cultured mouse brain slices;

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

[0009] (3) Cultivate the brain slices injected with glioma organoids obtained in step (2).

[0010] The co-culture model described in this application is a culture product obtained by culture.

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

[0012] Furthermore, the brain slice in step (1) is a telencephalon or brainstem slice.

[0013] Furthermore, the mouse brain slices obtained in step (1) were transferred onto the MF microporous membrane and cultured for more than one week.

[0014] Furthermore, 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 glioma organoids of WHO grade I-IV pathological type derived from patient tissues.

[0016] Furthermore, after using a pipette to suck up the glioma organoids in step (2), the pipette is inserted into the mouse brain slice at a 45° angle and slowly inserted to ensure that the glioma organoids do not escape before being withdrawn.

[0017] Furthermore, in step (3), the cells are first cultured for 48 hours using a medium comprising 2 mmol / L of L-glutamine substitute, 1×N2 additive, 1×B27 additive, 500 μmol / L of β-mercaptoethanol, 100 μmol / L of non-essential amino acids, 100×ITS1X, 100 U / mL penicillin, 100 μg / mL streptomycin, 20 ng / mL of EGF, and 20 ng / mL of bFGF, and then cultured for 1-2 weeks using a DMEM high-glucose medium comprising 20 ng / mL of EGF, 20 ng / mL of bFGF, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 1×N2 additive, and 1×B27 additive.

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

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

[0020] The so-called invasive phenotype refers to the ability of glioma tumors to invade normal tissues and undergo local infiltration and migration. The evaluation method specifically involves pathologically staining the co-culture model to characterize human tumor cells, and calculating the degree of migration of tumor cells away from the tumor center to measure the invasiveness of glioma organoids.

[0021] Specifically, the co-culture model was fixed with 4% paraformaldehyde overnight, dehydrated and embedded in sections, and HE staining and STEM121 human marker staining were completed.

[0022] Select human tumor cells labeled in at least five directions in each of at least three slices (or three co-culture model operations). Use Image J software to fit a circle to the main part of the glioma organoid, mark the center and outline of the circle, and measure the distance L from each selected human tumor cell to the center of the marked circle using Image J software. Measure the radius R of the marked circle using the following calculation formula:

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

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

[0025] In the test or screening, drug sensitivity is tested in a co-culture model, and the sensitivity of the drug to tumor cells and normal cells is observed simultaneously.

[0026] Specifically, the target drug intervention is applied in the culture medium and the drug can be administered repeatedly. After the drug intervention is completed, the cells are fixed with 4% paraformaldehyde overnight, dehydrated and embedded in sections, and HE staining or immunofluorescence staining is performed to observe the drug killing of tumor and normal mouse brain cells, and to detect drug sensitivity and safety.

[0027] On the other hand, the present application provides a mouse brain slice culture medium, which is a DMEM high-glucose medium including 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, the present application provides a buffered culture medium, which is a culture medium comprising 2 mmol / L of L-glutamine substitute, 1×N2 additive, 1×B27 additive, 500 μmol / L of β-mercaptoethanol, 100 μmol / L of non-essential amino acids, 100×ITS1X, 100 U / mL penicillin, 100 μg / mL streptomycin, EGF 20 ng / mL, bFGF 20 ng / mL, and a mixture of equal amounts of Neurobasal culture medium and DMEM / F12 culture medium.

[0029] The present invention simplifies and optimizes the mouse brain slice culture method, uses a dedicated culture medium and a mouse brain cutting mold, and efficiently produces sustainably cultured mouse brain slices in vitro. It also 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 assess drug sensitivity to glioma organoids and normal brain tissue with high efficiency and accuracy. Therefore, the present invention provides a new experimental platform and method for glioma invasiveness research and preclinical drug testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of the simplified mouse brain slice, brain glioma organoid and mouse brain slice co-culture model cultivation method of the present invention.

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

[0032] Figure 3 This is the pathological staining result of the co-culture model of brain glioma organoids and mouse brain slices established in the present invention.

[0033] Figure 4 This is an example of the method and results for evaluating the in vitro invasive phenotype of glioma organoids provided by the present invention;

[0034] Figure 5 This is the result of the in vitro preclinical drug testing method for glioma based on the above co-culture model provided by the present invention. DETAILED DESCRIPTION

[0035] Example 1 Mouse brain slice culture method:

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

[0037] (2) According to the present invention, a simplified method for culturing mouse brain slices in vitro is as follows:

[0038] I. Feeding female mice (Speff, ICR strain) and observing them daily. After 7 days of birth (can be extended to 14 days), six newborn mice were anesthetized on ice (7 days) or with an intraperitoneal injection of 50-150 mg / kg ketamine (8-14 days) according to their age.

[0039] II. After satisfactory anesthesia, the rat's head was cut with a large tissue snip and quickly rinsed in pre-chilled 75% medical alcohol (Anjie High-Tech). After removal, the head was thoroughly rinsed in pre-chilled artificial cerebrospinal fluid (Saiyintu, SSL6630) to remove the alcohol.

[0040] III. Use sterile forceps and ophthalmic scissors to separate soft tissue and the thin, soft skull, exposing the telencephalon and brainstem. Dissect and sever the olfactory nerves connecting to the telencephalon and the cranial nerves emanating from the brainstem to obtain intact mouse brain tissue, which is then temporarily stored in pre-chilled artificial cerebrospinal fluid.

[0041] IV. Prepare the blade in advance and sterilize it in pre-chilled 75% medical alcohol. Then temporarily place it in pre-chilled artificial cerebrospinal fluid.

[0042] V. Place the dissected mouse brain in a pre-autoclaved mouse brain cutting mold, ventral side down. Insert two blades into adjacent gaps in the brain cutting mold and cut. Remove the cephalad blade, removing the cephalad region of brain tissue as discarded material. Remove the caudal blade, attaching the target brain slice to the cephalad side of the caudal blade.

[0043] VI. Cut three slices in a row. After removing the cranial blade, immediately place it in the next cutting die gap just behind the caudal blade. Repeat the above steps to obtain three slices.

[0044] VII. After slicing, rinse the brain slices with pre-chilled artificial cerebrospinal fluid (ACS) into a large dish containing pre-chilled ACS. Once all slices are removed, carefully transfer the slices to a MF microporous membrane (Millipore, PICM03050) using sterile blunt-tipped forceps, three slices per membrane, and place them in a six-well plate.

[0045] VIII. Add 1.2-1.5 mL of specialized culture medium to each well and continue culturing in a 37°C 5% CO2 incubator.

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

[0047] X. HE staining and multiple immunofluorescence staining of cultured mouse brain slices ( Figure 2 ).

[0048] Example 2 Co-culture Model Cultivation Method of Glioma Organoids and Mouse Brain Slices

[0049] 1. Culturing mouse brain slices in vitro using the method in Example 1;

[0050] II. Cultured glioma organoids (derived from a patient with pilocytic astrocytoma, CNS WHO grade I, approximately 200 μm in diameter) were aspirated using a minimal-gauge pipette tip (with the tip partially trimmed). Carefully inserted the organoids into the mouse brain slice at an angle of approximately 45°. Slowly insert the organoids until they are fully dislodged.

[0051] III. Use buffered culture medium to replace the culture medium, which is prepared according to the following composition: Neurobasal Medium 250 mL (Gibco, 21103049), DMEM / F12 medium 250 mL (Cipro, C11330500BT), L-glutamine substitute 2 mol / L (Cipro, S60314014A), 1× N2 supplement (50×, Cipro S60314017A), 1× B27 supplement (50×, Cipro S60314015A), β-mercaptoethanol 500 μmol / L (Cipro, S60314019A), non-essential amino acids 100 μmol / L (Cipro, S60314011A), 100× ITS1X (Cipro, SC25800), 100 U / mL penicillin and 100 μg / mL streptomycin (100×, Thermo Fisher 10378016), EGF 20 ng / mL (Ceintu SC102), bFGF 20 ng / mL (Ceintu SC107-10 μg).

[0052] IV. After 48 hours, the slices were cultured in a 37°C 5% CO2 incubator. The culture medium was changed every 3 days. After 1-2 weeks, the invasion phenotype evaluation or drug testing experiments were performed. Figure 1 ).

[0053] Example 3-4 More methods for co-culture of glioma organoids and mouse brain slices

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

[0055] HE and multiple immunofluorescence staining were performed on the organoid-brain slice co-culture model and the source organoids and parent tissues cultured in Examples 2-4. The co-culture model retained the complexity of the tumor microenvironment and more realistically simulated the growth characteristics of tumors in vivo ( Figure 3 ).

[0056] Example 5 Method for evaluating the invasive phenotype of glioma organoids in vitro

[0057] I. Using the organoid and mouse brain slice co-culture model established in Examples 2 to 4 of the present invention;

[0058] II. After one week of culture, the cells were fixed, dehydrated, embedded, and paraffin-sectioned. Hematoxylin and eosin staining was performed, and the degree of tumor cell migration away from the tumor center was calculated in five directions in each of at least three sections (or three co-culture models) to measure the invasiveness of the glioma organoids.

[0059] III. Using Image J software, we fitted a circle to the main body of the glioma organoid, marking the center and outline of the circle. We also measured the distance (L) from each selected human tumor cell to the center of the marked circle in each direction using Image J software. The radius (R) of the marked circle was also measured. The invasiveness score was calculated using the formula: AVG (average) [(LR) / R] × 100%.

[0060] IV. The invasive phenotype of glioma organoids measured by the co-culture model of organoids and mouse brain slices established in Examples 2-4 Figure 4 As shown, the most malignant diffuse midline glioma (the glioma organoids in Example 4 were derived from a diffuse midline glioma with H3K27M mutation) had the highest degree of invasion ( Figure 4 ).

[0061] Example 6 Co-culture Model for Glioma Preclinical Drug Testing in Vitro

[0062] I. Using the co-culture model of organoids and mouse brain slices established by the present invention;

[0063] II. Add the target concentration of drug (e.g., 10 nM Panobinostat) to the brain slice culture medium and continue culturing in a 37°C 5% CO2 incubator;

[0064] III. After 1 week of continuous culture, the cells were fixed, dehydrated, embedded, and paraffin-sectioned. HE staining was performed to observe the growth of tumor organoids and the morphology of mouse brain cells to test the efficacy and safety of the drug. Figure 5 As shown, Panobinostat is a potentially effective drug, and the tumor at the organoid inoculation site (center) disappears and a cavity is formed.

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) obtaining mouse brain slices, culturing them, and obtaining cultured mouse brain slices; (2) injecting the glioma organoids into the cultured mouse brain slices obtained in step (1) to obtain brain slices injected with glioma organoids; (3) Cultivating the brain slice injected with the glioma organoid obtained in step (2) to obtain the co-culture model.

2. The co-culture model according to claim 1, wherein the mice in step (1) are ICR newborn mice 1-2 weeks old; the brain slices are telencephalon or brainstem slices; the mouse brain slices are transferred to the MF microporous membrane after being taken; and the cells are cultured in 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 for more than 1 week.

3. The co-culture model according to claim 1 or 2, wherein the glioma organoids in step (2) are glioma organoids of WHO grade I-IV pathological type derived from patient tissue; after the glioma organoids are aspirated with a pipette, they are inserted into the mouse brain slice at a 45° angle and slowly inserted to ensure that the glioma organoids do not escape before being withdrawn.

4. The co-culture model according to any one of claims 1 to 3, wherein in step (3), the cells are first cultured for 48 hours in a medium prepared by mixing equal amounts of Neurobasal medium and DMEM / F12 medium, comprising 2 mol / L L-glutamine substitute, 1×N2 additive, 1×B27 additive, 500 μmol / L β-mercaptoethanol, 100 μmol / L non-essential amino acids, 100×ITS1X, 100 U / mL penicillin, 100 μg / mL streptomycin, 20 ng / mL EGF, and 20 ng / mL bFGF; and then cultured for 1-2 weeks in 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.

5. A method for preparing a co-culture model of glioma organoids and mouse brain slices, characterized in that: The method comprises: (1) obtaining mouse brain slices, culturing them, and obtaining cultured mouse brain slices; (2) injecting the glioma organoids into the cultured mouse brain slices obtained in step (1) to obtain brain slices injected with glioma organoids; (3) Cultivating the brain slice injected with the glioma organoid obtained in step (2) to obtain the co-culture model.

6. The method according to claim 5, wherein: The mice in step (1) are ICR newborn mice of the 1-2 week old strain; the brain slices are telencephalon or brainstem slices; the mouse brain slices are transferred onto MF microporous membranes after being taken; and the cells are cultured for more than 1 week using DMEM high-glucose medium containing 20 ng / mL EGF, 20 ng / mL bFGF, 100 U / mL penicillin, 100 μg / mL streptomycin, 1×N2 supplement, and 1×B27 supplement; The glioma organoids in step (2) are glioma organoids of WHO grade I-IV pathological type derived from patient tissue; After using a pipette to pick up the glioma organoids, insert them into the mouse brain slice at a 45° angle and slowly push them in to ensure that the glioma organoids do not escape before withdrawing them. In step (3), the cells are first cultured for 48 hours using a medium containing 2 mmol / L of L-glutamine substitute, 1×N2 additive, 1×B27 additive, 500 μmol / L of β-mercaptoethanol, 100 μmol / L of non-essential amino acids, 100×ITS1X, 100 U / mL penicillin, 100 μg / mL streptomycin, 20 ng / mL of EGF, and 20 ng / mL of bFGF, and then cultured for 1-2 weeks using a DMEM high-glucose medium containing 20 ng / mL of EGF, 20 ng / mL of bFGF, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 1×N2 additive, and 1×B27 additive.

7. Use of the co-culture model according to any one of claims 1 to 4 in evaluating the invasive phenotype of glioma organoids in vitro.

8. The use according to claim 7, wherein the co-culture model is fixed with 4% paraformaldehyde overnight, dehydrated, embedded, and sliced, and stained with HE and STEM121 human markers; at least five directional labeled human tumor cells are selected from each of at least three slices, and a circle is fitted to the main part of the glioma organoid using Image J software, with the center and outer contour of the circle marked. The distance L from each selected human tumor cell to the center of the marked circle is measured using Image J software, and the radius R of the marked circle is measured. The invasiveness is calculated using the formula: invasiveness = [(LR) / R] × 100%.

9. Use of the co-culture model according to any one of claims 1 to 4 in in vitro drug testing or screening of gliomas.

10. The use according to claim 9, wherein the target drug intervention is applied to the culture medium of the co-culture model; after the drug intervention, the cells are fixed with 4% paraformaldehyde overnight, dehydrated and embedded in sections, and stained with HE or immunofluorescence; the drug-induced damage to tumor and normal mouse brain cells is observed, and the drug sensitivity and safety are tested.

Images