Myeloblastoma organoid as well as culture medium, culture method and application thereof

Abstract

The invention belongs to the technical field of tumor organoid culture, and particularly relates to a myeloblastoma organoid as well as a culture medium, a culture method and application thereof. The culture medium is prepared from DMEM (Dulbecco Modified Eagle Medium): F12, Neurobase, KOSR (Kovary Orthogonal Sequence Repeat), GlutaMax, A-A, CDLC (Clinical Dissolved Liquid Chromatography), N2, B27, Heparin, T3 and BDNF. The culture method comprises the following steps: taking a medulloblastoma tumor tissue, directly cutting into blocks, and then carrying out red blood cell lysis to obtain small tumor blocks; the myeloblastoma tumor tissue does not need to be subjected to enzyme digestion treatment; the small tumor blocks are cultured with the culture medium, and myeloblastoma organoid is generated; and carrying out subculture on the obtained myeloblastoma organoid by adopting the culture medium to obtain the myeloblastoma organoid. The myeloblastoma organoid obtained through culture highly retains the tissue structure and the cell type of a parent tumor, can simulate in-vivo growth and development of the tumor in vitro, and can be used for personalized medicine screening.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor organoid culture, and specifically relates to a medulloblastoma organoid and a culture medium, a culture method and an application thereof. Background Art

[0002] Medulloblastoma is one of the most common brain tumors in children and is highly heterogeneous. Medulloblastoma organoids are an in vitro three-dimensional cell culture model for studying medulloblastoma. They are three-dimensional tissues generated from patient tumor cells that recapitulate the characteristics of the parent tumor. Qualified tumor organoid models can maintain cell diversity, maintain interactions between native cells, recapitulate defined histological features, and maintain the transcriptome and mutation spectrum of the parent tumor. They are important tools for studying tumor occurrence, development, drug response, and personalized treatment.

[0003] There are cases of generating glioblastoma organoids (GBOs) from surgically removed patient tumor tissue using chemically defined culture media, which maintain the cellular heterogeneity of the parental tumor by maintaining cell-cell interactions and extremely small clonal fragments. The culture medium is composed of DMEM:F12, Neurobasal, GlutaMax (100X), NEAAs (100X), PenStrep (100X), N2 (100X), B27 w / o vitaminA (50X), Insulin (2.5mg / mL) and 2-Me (1:1000). The formula idea is derived from the improvement of the cortical development medium of brain organoids. For details, see "Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing" and "A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter-and Intra-tumoral Heterogeneity" published by Fadi Jacob et al. However, the existing method of cryopreservation of glioblastoma organoids is unstable after thawing, and this method is only applicable to glioblastoma organoids. Medulloblastoma often occurs in the cerebellum, the tumor is sparse, and primary cells mostly grow in suspension. The current organoid culture method and culture medium formula are not suitable for the establishment of a medulloblastoma organoid model that is highly similar to tumor tissue, and both the culture method and culture medium formula need to be improved.

[0004] In the prior art, patent CN116769715A discloses a culture medium and a culture method for medulloblastoma organoids and 3D microsphere suspension culture, wherein the culture medium is composed of the following components: basal culture medium, B27 without vitamin A, N2, N-acetylcysteine, FGFb, Noggin, R-spondin, Wnt3a, CHIR99021, Laminin, Sialicacid, NEAA, a penicillin-streptomycin mixture and Primocin; the basal culture medium is composed of Advanced DMEM / F12 and NeuralBasal culture medium mixed in a volume ratio of 1:10-150:1. The suspension culture method of medulloblastoma organoids and 3D microspheres includes the following steps: (1) isolating and obtaining medulloblastoma cells; the medulloblastoma cells are obtained by pre-treatment of surgical resection specimens or biopsy tissues of medulloblastoma; (2) resuspending the cells using the above-mentioned culture medium, transferring them to ultra-low adhesion cell culture dishes or culture plates, and culturing them in a carbon dioxide incubator until medulloblastoma organoids or medulloblastoma 3D microspheres are obtained. The patent uses a mixed enzyme solution of trypsin, collagenase, and DNAse to digest the tissue and then recombine it to generate medulloblastoma sphere organoids. This organoid establishment method digests the tumor tissue into single cells, and then randomly recombines to form a suspension culture 3D sphere containing multiple cells. This type of organ destroys the unique tumor structure of medulloblastoma (such as the pseudorosette structure in a certain medulloblastoma subtype), and also destroys the interaction between cells. The regenerated medulloblastoma changes the tumor environment, has no vascular structure, and cannot well simulate the morphological characteristics of medulloblastoma in vivo.

[0005] Medulloblastomas exhibit extensive inter- and intratumor heterogeneity, yet most in vitro models fail to retain the cellular and mutational diversity of the parental tumors and often require long generation times and carry variable rates, leaving a vacancy in organoid models of pediatric malignant brain tumors. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a culture medium for culturing medulloblastoma organoids. The culture medium can better retain the tissue structure and cell type of the parent tumor by culturing medulloblastoma organoids.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A culture medium for culturing medulloblastoma organoids, wherein the organoids to be cultured are medulloblastoma organoids; the culture medium comprises the following components: a basal culture medium, KOSR with a final concentration of 1%-10%, GlutaMax with a final concentration of 1X, AA with a final concentration of 1X, CDLC with a final concentration of 1%-3%, N2 with a final concentration of 1X, and B27 with a final concentration of 1X.

[0009] Preferably, the final concentration of the KOSR is 2%; and the final concentration of the CDLC is 1%.

[0010] Furthermore, the basic culture medium includes DMEM:F12 and Neurobasal; the volume ratio of DMEM:F12 to Neurobasal is 1-5:1-5.

[0011] Preferably, the volume ratio of DMEM:F12 and Neurobasal is 1:1.

[0012] Furthermore, the culture medium also contains Heparin with a final concentration of 3 to 8 μg / mL, T3 with a final concentration of 0.3 to 0.8 ng / mL, and BDNF with a final concentration of 10 to 20 ng / mL.

[0013] Preferably, in the culture medium, the final concentration of Heparin is 5 μg / mL, the final concentration of T3 is 0.5 ng / mL, and the final concentration of BDNF is 14 ng / mL.

[0014] Most preferably, the culture medium consists of the following components: DMEM:F12, Neurobasal, KOSR with a final concentration of 2%, GlutaMax with a final concentration of 1X, AA with a final concentration of 1X, CDLC with a final concentration of 1%, N2 with a final concentration of 1X, B27 with a final concentration of 1X, Heparin with a final concentration of 5 μg / mL, T3 with a final concentration of 0.5 ng / mL and BDNF with a final concentration of 14 ng / mL; the volume ratio of the DMEM:F12 and Neurobasal is 1:1.

[0015] A second object of the present invention is to provide a method for culturing medulloblastoma organoids without enzymatic digestion.

[0016] To achieve the above object, the present invention adopts the following technical solutions:

[0017] A method for culturing medulloblastoma organoids without enzymatic digestion, comprising the following steps:

[0018] (1) taking medulloblastoma tumor tissue, directly cutting it into pieces and then performing red blood cell lysis to obtain small tumor pieces; the medulloblastoma tumor tissue does not need to be subjected to enzyme digestion treatment;

[0019] (2) culturing the small tumor mass obtained in step (1) using the aforementioned culture medium to generate medulloblastoma organoids;

[0020] (3) The medulloblastoma organoids obtained in step (2) are subcultured using the aforementioned culture medium to obtain medulloblastoma organoids.

[0021] Furthermore, in step (1), the medulloblastoma tumor tissue is preferably medulloblastoma tumor tissue removed by surgery.

[0022] Furthermore, in step (1), the medulloblastoma tumor tissue is first washed with ice-cold PBS and triple antibodies, and then cut into pieces.

[0023] Furthermore, in step (1), the red blood cell lysis comprises: adding red blood cell lysis buffer and incubating on a shaker at a rotation speed of 5 rpm for 5-15 minutes.

[0024] Furthermore, in step (2) and step (3), the culture conditions are: culturing on a horizontal shaker in a cell culture incubator at 37±5°C, 5±2% CO2, and 90±5% relative humidity, with the rotation speed set to 80±5 rpm on the first day and to 100±5 rpm in the later period.

[0025] Furthermore, in step (2) and step (3), 10-20 tumor slices are added to 4 mL of medulloblastoma organoid culture medium for culture.

[0026] Preferably, the method specifically comprises the following steps:

[0027] ① Tissue processing: Take the medulloblastoma tumor tissue removed during surgery, and wash it with ice-cold PBS and three antibodies; cut the washed tumor tissue into small pieces of 0.5-1 mm in a glass dish placed on ice, wash it with room temperature PBS, add red blood cell lysis buffer, and incubate it on a shaker at a speed of 5 rpm for 5-15 minutes; wash it with room temperature DMEM:F12 to obtain small tumor pieces.

[0028] ② Tissue block culture: The small tumor block obtained in step ① is cultured using the aforementioned culture medium on a horizontal shaker in a cell culture incubator at 37°C, 5% CO2, and 90% relative humidity; the rotation speed is set to 80 rpm on the first day and to 100 rpm in the later period; the culture medium is replaced every 3 days to obtain organoids.

[0029] ③ Medulloblastoma organoid passaging: The medulloblastoma organoids are passaged when their diameter reaches about 1-2 mm. Passage is performed at room temperature in a glass dish in a clean bench using a dissecting microscope. Use dissecting scissors to cut the center of each organoid into four equal pieces, and use the culture medium mentioned above for culturing medulloblastoma organoids, and culture on a horizontal shaker in a cell culture incubator at 37°C, 5% CO2, and 90% relative humidity; set the speed to 80 rpm on the first day and 100 rpm in the later period. After one week of organoid passaging, wait until the periphery of the organoid is rounded before freezing.

[0030] Furthermore, the medulloblastoma organoids are cryopreserved by the following method: the medulloblastoma organoids are incubated with a medulloblastoma organoid culture medium containing 8-15 μM Y-27632, and then cryopreserved with a freezing solution; the freezing solution consists of a medulloblastoma organoid culture medium, Y-27632 and DMSO; the content of Y-27632 in the freezing solution is 8-15 μM, and the content of DMSO is 10%.

[0031] Preferably, the medulloblastoma organoids are incubated with a medulloblastoma organoid culture medium containing 10 μM Y-27632 and then cryopreserved with a freezing solution; the freezing solution consists of a medulloblastoma organoid culture medium, Y-27632 and DMSO; the content of Y-27632 in the freezing solution is 10 μM, and the content of DMSO is 10%.

[0032] Preferably, the freezing method specifically comprises the following steps:

[0033] Replace the organoid culture medium with fresh medium containing 10μM Y-27632; incubate the tumor slices at 100 rpm for 3 hours; culture in a cell culture incubator at 37°C, 5% CO2, and 90% relative humidity; discard the organoid culture medium containing 10μM Y-27632, add room temperature organoid freezing solution, and incubate at room temperature at 5 rpm for 5 minutes; discard the freezing solution, add freezing solution pre-cooled at 4°C to resuspend the organoids; transfer to cryopreservation tubes, gradient freeze in a -80°C refrigerator for 24 hours, and transfer to liquid nitrogen for long-term storage.

[0034] Furthermore, the medulloblastoma organoids were revived by the following method: after thawing the frozen medulloblastoma organoids, medulloblastoma organoid culture medium containing 8-15 μM Y-27632 and the aforementioned freezing solution were added for overnight static culture; on the next day, culture was carried out on a horizontal shaker using a culture medium without 8-15 μM Y-27632.

[0035] Preferably, after the frozen medulloblastoma organoids are thawed, medulloblastoma organoid culture medium containing 10 μM Y-27632 and the aforementioned freezing solution are added for overnight static culture; the next day, culture is carried out on a horizontal shaker using a culture medium without 10 μM Y-27632.

[0036] Preferably, the resuscitation method specifically comprises the following steps:

[0037] The organoids were taken out of liquid nitrogen, and after thawing, medulloblastoma organoid culture medium containing 10 μM Y-27632 was added, and the freezing solution containing DMSO was slowly diluted while shaking; the supernatant was discarded, medulloblastoma organoid culture medium containing 10 μM Y-27632 was added and transferred to the culture plate, and cultured overnight in a sterile incubator at 37°C, 5% CO2, and 90% relative humidity; on the next day, the culture medium was replaced with medulloblastoma organoid culture medium without 10 μM Y-27632, and the culture plate was transferred to a 100 rpm horizontal shaker and cultured in a sterile incubator at 37°C, 5% CO2, and 90% relative humidity.

[0038] The third object of the present invention is to provide a medulloblastoma organoid cultured using the aforementioned method.

[0039] To achieve the above object, the present invention adopts the following technical solutions:

[0040] Medulloblastoma organoids cultured using the aforementioned method.

[0041] Furthermore, the medulloblastoma organoids contain the histological structure of the parent tumor.

[0042] Furthermore, the medulloblastoma organoids retain the neuroblastoma rosette or pseudorosette structure of the parental tumor; and / or retain the cell types of the parental tumor: β3-tubulin (neurons), GFAP (astroglia), iba1 (microglia), olig2 (oligodendrocytes) and CD31 (endothelial cells - representing the vascular system), and keep their distribution characteristics unchanged.

[0043] A fourth object of the present invention is to provide a use of the aforementioned medulloblastoma organoid in the preparation of drugs for screening anti-medulloblastoma and / or constructing a medulloblastoma animal model.

[0044] The beneficial effects of the present invention are:

[0045] 1. The present invention adopts a culture medium formula for the stable growth period of cerebellar development of brain organoids, combined with the nutritional characteristics required by medulloblastoma cell types, to improve it into a culture medium suitable for the growth of medulloblastoma organoids. After a lot of research, the present invention found that medulloblastoma organoids do not need unclear additives such as serum and extracellular matrix (such as Matrigel) to survive and form high-quality organoids; the culture medium has clear ingredients and does not contain serum, bFGF and Matrigel matrix glue, etc., which contain unknown ingredients that may cause cell differentiation. The culture medium contains various nutrients that are beneficial to the growth of various types of cells in medulloblastoma tissues. After multiple adjustments to the formula and ratio, an optimal ratio is achieved.

[0046] 2. The present invention uses a culture medium with a defined chemical composition, and brain tumor organoids can be cultured directly from surgically removed tumor tissue of patients without the need to decompose cells.

[0047] 3. The current medulloblastoma organoid culture method is to use a mixed enzyme solution of trypsin, collagenase, and DNAse to digest the tissue and then recombine it to generate medulloblastoma tumor sphere organoids. The organoid culture method of the present invention does not undergo enzymatic digestion, and generates organoids that are highly consistent with the parent tumor without destroying the internal environment homeostasis and cell interaction of the original tumor. Such organs can simulate the growth and development of tumors in vivo in vitro, can be used for personalized drug screening, and can be pathologically sectioned and pathologically stained like tumor tissue blocks. It is an organoid model that highly fits in vivo tumors.

[0048] 4. The organoids cultured with the culture medium of the present invention highly retain the tissue structure of the parental tumor, such as neuroblastoma rosettes or pseudorosettes in some cases; at the same time, the proliferation characteristics (Ki67) and cell types of the parental tumor are also retained, including neurons (β3-tubulin), astrocytes (GFAP), microglia (iba1), oligodendrocytes (olig2) and endothelial cells (CD31), etc. The present invention provides support for the occurrence, development, drug response and personalized treatment of medulloblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Flow chart for the culture of medulloblastoma organoids;

[0050] Figure 2 This is a picture of the medulloblastoma organoid culture process, in which: Figure 2 -A is a picture of medulloblastoma tumor tissue removed during surgery before and after cleaning; Figure 2 -B is a picture of a small tumor piece cut from medulloblastoma tumor tissue; Figure 2-C is the image of medulloblastoma organoid culture (CBOs) and medulloblastoma organoid culture (CBOs) after recovery, the scale bar is 500μm; Figure 2 -D is the area statistics of medulloblastoma organoids at 4 weeks of culture (n=5-6);

[0051] Figure 3 The results of HE staining for histological analysis of medulloblastoma organoids, CD31 marking vasculature, Iba1 marking microglia, Olig2 marking oligodendrocytes, and Ki67 marking proliferating cells, with a scale bar of 50 μm;

[0052] Figure 4 This is the result of the identification of medulloblastoma organoids using GFAP to label astrocytes and β3-Tubulin to label neurons. The scale bar is 50 μm.

[0053] Figure 5 Illustration of the organizational structure of medulloblastoma organoids. The white arrow indicates that the organoids retain the pseudorosette structure of the parental tumor. DETAILED DESCRIPTION

[0054] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0055] In the embodiment of the present invention, the medulloblastoma tissue was obtained from the Department of Neurosurgery of the Children's Hospital Affiliated to Chongqing Medical University, and has been approved by the Ethics Committee of the Children's Hospital Affiliated to Chongqing Medical University, with approval number: (2019) Lun Shen (Clinical Research) No. (16); and the samples were processed in strict accordance with the requirements of the Ethics Committee of the Children's Hospital Affiliated to Chongqing Medical University; at the same time, the patient's informed consent was obtained.

[0056] In the examples of the present invention, the basic information of the main reagents involved is shown in Table 1.

[0057] Table 1. Reagent information list

[0058]

[0059]

[0060] In the embodiment of the present invention, the triple antibiotic is an antibiotic-antifungal agent, namely AA (100X) in Table 1.

[0061] In the present invention, the culture medium for culturing medulloblastoma organoids is referred to as medulloblastoma organoid culture medium. In the culture medium, DMEM:F12 and Neurobasal are the basic culture medium components, and together with KOSR, GlutaMax, AA, CDLC, N2, and B27, they constitute the main components of the medulloblastoma organoid culture medium, which are used to maintain the growth of brain tissue cells and tumor cells. Heparin, T3, and BDNF are also added to the medulloblastoma organoid culture medium. These three components are additives that promote the activity of various types of medulloblastoma cells and play an auxiliary role.

[0062] DMEM / F12 medium: DMEM medium has a high concentration of nutrients, while F12 medium has a complex composition and contains a variety of trace elements. DMEM and F12 are combined in a 1:1 ratio, called DMEM / F12 medium. This medium is suitable for cell culture under conditions of low serum content and is often used for cell cloning and stem cell culture.

[0063] Neurobasal is a culture medium designed specifically for neural cells and is suitable for the culture of neurons and glial cells.

[0064] KOSR, or KnockOut Serum Replacement, is a serum substitute that is particularly suitable for certain cell types that are sensitive to serum components, such as stem cells and certain primary cells. It has the following characteristics in cell culture: KOSR contains a variety of growth factors, hormones and nutrients that can support the growth and proliferation of a variety of cell types; compared with traditional fetal bovine serum, KOSR has a lower level of endotoxin, which helps reduce the risk of contamination in cell culture; KOSR does not contain animal-derived components and is suitable for cell culture that needs to avoid animal-derived contamination, such as the production of cells and proteins for clinical applications; due to the consistency of its ingredients, KOSR helps to improve the repeatability and reliability of cell culture experiments.

[0065] GlutaMax is a dipeptide form of L-glutamine (L-alanyl-L-glutamine), which is extremely stable in aqueous solution and does not spontaneously degrade during long-term storage and culture, reducing the generation of ammonia, thereby improving the stability of the culture medium, minimizing the accumulation of toxic components of ammonia, and optimizing cell performance. L-glutamine is an essential amino acid for cell culture and is involved in the formation of other amino acids, nucleic acid and protein synthesis, and glucose production.

[0066] AA is an antibiotic-antimycotic used to prevent bacterial and fungal contamination. The solution contains 10,000 units / mL penicillin, 10,000 μg / mL streptomycin, and 25 μg / mL Gibco amphotericin B. The antibiotics penicillin and streptomycin have a combined effective antimicrobial effect against gram-positive and gram-negative bacteria and are used to prevent bacterial contamination of cell cultures. Amphotericin B inhibits multicellular fungi and yeasts, thereby preventing fungal contamination of cell cultures.

[0067] CDLC is a lipid concentrate with clear chemical composition. Lipids are important components of cell membranes and are used to reduce or replace fetal bovine serum in cell culture media. They can be used for the growth and maintenance of a variety of cells.

[0068] N2: N-2 Supplement is a chemically defined serum-free supplement based on Bottenstein's N-1 formulation. It can be used for low-density or high-density growth and short-term or long-term survival of embryonic, postnatal, and adult hippocampal and other CNS neurons. N-2 Supplement is provided as a 100X concentrate and can be used with Neurobasal medium or with DMEM.

[0069] B27: B-27 Supplement is a serum-free supplement optimized for use with Neurobasal Medium for culturing neuronal cells without the need for an astrocyte feeder layer.

[0070] Heparin: In the presence of heparin, it can increase the proliferation of vascular endothelial cells and increase cell viability.

[0071] T3 is a biologically active thyroid hormone, also known as 3,3′5-triiodo-L-thyronine. T3 promotes the metabolic breakdown of glucose, fat, and protein by increasing the levels of many metabolic enzymes, such as hexokinase for oxidative phosphorylation, hepatic glucose-6-phosphatase, and mitochondrial enzymes.

[0072] BDNF is a member of the neurotrophic factor family. It can support the survival, development, differentiation and repair of various neurons, participate in the regulation of neuronal structure and morphological plasticity, and also participate in synaptic connection and neurotransmitter transmission.

[0073] Medulloblastoma is often divided into 4 different molecular subtypes: SHH type, Wnt type, type 3 and type 4. Studies have shown that SHH type medulloblastoma originates from cerebellar granule cell progenitors; type 3 tumors may originate from early cerebellar progenitors; Wnt type tumors may originate from multipotent progenitors of the lower rhomboid lip in the dorsal brainstem; type 4 tumors may originate from glutamatergic progenitors, which may be unipolar brush cells in the cerebellum. Studies have shown that these progenitor cells progress to medulloblastoma when they are continuously stimulated or fail to exit the cell cycle. This organoid culture medium is rich in nutrients and is designed for medulloblastoma organoid culture based on the cell type and characteristics of medulloblastoma. It can support the proliferation and growth of tumor stem cells, neurons and glial cells in medulloblastoma organoids.

[0074] In the embodiment of the present invention, the medullary organoid culture medium is prepared as follows: according to the formula shown in Table 2, DMEM: F12 250 mL, Neurobasal 250 mL, KOSR 10 mL, GlutaMax (100X) 5 mL, AA (100X) 5 mL, CDLC

[0075] 5mL, N2 (100X) 5mL, B27 (50X) 10mL, Heparin 2.5mg, T3 0.25mg, BDNF 7mg, prepare the medulloblastoid culture medium for subsequent experiments.

[0076] Example 1. Culture medium for culturing medulloblastoma organoids

[0077] The recipe for the culture medium used to culture medulloblastoma organoids is shown in Table 2.

[0078] Table 2. Medulloblastoma organoid culture medium formula

[0079]

[0080] Among them, DMEM:F12 and Neurobasal are the components of the basal culture medium, and the preferred ratio of the two is 1:1; KOSR is a liquid, and the final concentration of addition is 2%; GlutaMax is a 100X concentrated solution, and the required final concentration is 1X; AA is a 100X concentrated solution, and the required final concentration is 1X; CDLC is a liquid, and the final concentration of addition is 1%; N2 is a 100X concentrated solution, and the required final concentration is 1X; B27 is a 50X concentrated solution, and the required final concentration is 1X; the required final concentration of Heparin is 5μg / mL; the required final concentration of T3 is 0.5ng / mL, and the required final concentration of BDNF is 14ng / mL.

[0081] Example 2. Method for culturing medulloblastoma organoids

[0082] The medulloblastoma organoid culture medium of Example 1 was used to culture medulloblastoma organoids. The culture process is shown in Figure 1-2 , specifically including the following steps:

[0083] (1) Tissue processing

[0084] Medulloblastoma tumor tissue removed during surgery was immediately transferred to a 50mL centrifuge tube containing 10mL frozen tissue preservation solution for ice transportation and short-term storage, and the next step was processed within 2 hours. After arriving at the laboratory, the tumor tissue was washed three times with 10mL of ice PBS and three antibodies in the clean bench. The tissue was cut into small pieces of 0.5-1 mm in a glass dish placed on ice, washed three times with room temperature PBS, added with 10-20mL of red blood cell lysis buffer, and the centrifuge tube was placed on a shaker, set to 5 rpm, and incubated at room temperature for 5-15 minutes (the more red blood cells in the tumor block, the longer the incubation time), and washed three times with 10mL of room temperature DMEM: F12, allowing the tumor pieces to sink to the bottom of the centrifuge tube, and then carefully aspirated and discarded the supernatant to obtain small tumor blocks.

[0085] (2) Tissue explant culture (organoid generation)

[0086] Transfer small tumor pieces to a 6-well, flat-bottom, untreated culture plate, and take 10-20 tumor pieces and 4 mL of medulloblastoid culture medium per well. Culture on a horizontal shaker in a cell culture incubator at 37°C, 5% CO2, and 90% relative humidity. 80 rpm on the first day and 100 rpm in the later stages. Change the culture medium every 3 days. Organoids form round or nearly round shapes in two weeks, and organoids are generated. Remove the non-growing parts until they are completely composed of round GBOs.

[0087] (3) Passaging of medulloblastoma organoids

[0088] Medulloblastoma organoids are passaged when their diameter reaches about 1-2 mm to avoid massive necrotic cell death in the center. Passage is performed at room temperature in a glass dish in a clean bench using a dissecting microscope. Use dissecting scissors to cut the center of each organoid into four equal pieces, and passage them in fresh medulloblastoma organoid culture medium. Replace the new medulloblastoma organoid culture medium with 10-20 tumor slices per well on a 6-well, flat-bottomed, untreated culture plate and add 4 mL of medulloblastoma organoid culture medium for culture. Culture on a horizontal shaker in a cell culture incubator at 37°C, 5% CO2, and 90% relative humidity. The speed is 80 rpm on the first day and 100 rpm in the later stage. After one week of organoid passage, the organoid can be frozen after the periphery is rounded. See Passaging of Tumor Organoids for details. Figure 2 -A.

[0089] Example 2. Cryopreservation and thawing of medulloblastoma organoids

[0090] (1) Cryopreservation of medulloblastoma organoids

[0091] Organoids were replaced with fresh medulloblast organoid medium containing 10 μM Y-27632. Tumor pieces were incubated at 100 rpm for 3 hours. Incubate in a cell culture incubator at 37°C, 5% CO2, and 90% relative humidity. Incubation with 10 μM Y-27632 helps improve cell viability during cryopreservation. Aspirate the organoid medium containing 10 μM Y-27632, add 5 mL / well of room temperature organoid cryopreservation medium (medulloblast organoid medium with 10 μM Y-27632 and 10% DMSO), and incubate at room temperature at 5 rpm for 5 minutes to allow the cryopreservation medium to fully perfuse the organoids. Compared with single cell suspensions, organoids are large and need to be incubated in cryopreservation medium for a short period of time to allow the medium containing DMSO to better penetrate the cells. This incubation step helps improve cell viability during cryopreservation. Aspirate the cryopreservation medium and add 1 mL of new cryopreservation medium (pre-cooled at 4°C) to resuspend the organoids. Transfer the organoids and cryopreservation solution to cryopreservation tubes. Label the cryopreservation tubes with sample information. Place the cryopreservation tubes containing organoids in a gradient cell freezing box (freeze cells at a rate of -1°C / min) and quickly place the container in a -80°C freezer for 24 hours. Then transfer to liquid nitrogen for long-term storage.

[0092] (2) Organoid resuscitation

[0093] Remove the cryovial from liquid nitrogen and immediately thaw in a 37°C water bath, gently swirling to ensure even thawing, and remove the cryovial when only a small piece of ice remains. Transfer the organoid and cryopreservation solution to a 50 mL centrifuge tube in a clean hood, add 10 mL of myeloid organoid medium containing 10 μM Y-27632, and slowly dilute the cryopreservation solution containing DMSO while adding the cryopreservation solution. Aspirate the supernatant and add 4 mL / tube of myeloid organoid medium containing 10 μM Y-27632. Transfer the organoid to a 6-well, flat-bottom, untreated culture plate, 1 well per tube. The revived organoids are cultured in a sterile incubator at 37°C, 5% CO2, and 90% relative humidity overnight. The next day, the culture medium is replaced with myeloid organoid medium without 10 μM Y-27632, and the culture plate is transferred to a 100 rpm horizontal shaker and cultured in a sterile incubator at 37°C, 5% CO2, and 90% relative humidity. Organoids can be used for experiments after 2 weeks of recovery.

[0094] Example 3. Identification of medulloblastoma organoids

[0095] (1) Sampling medulloblastoma tissue and organoids for downstream analysis

[0096] Collect tumor tissue or organoids in a centrifuge tube, wash with ice PBS, add pre-cooled 4% formaldehyde solution and fix it upright at 4°C overnight. On the second day, add 30% sucrose solution and incubate upright at 4°C overnight to protect the tissue from low temperatures. On the third day, transfer the tumor or organoid to a cryoembedding mold and add tissue cryoembedding agent to completely fill the mold. Stir the embedding agent with a pipette tip to ensure that the tumor block or organoid is in the middle. Put the mold into -80°C to solidify and then store at -80°C.

[0097] (2) Immunofluorescence staining of medulloblastoma and organoids

[0098] After slicing according to the conventional frozen sectioning method, stick it to an adhesive slide. The thickness of the immunofluorescence staining section is 15μm, and the thickness of the HE staining section is 10μm. Dry in a 60℃ oven for 10-20 minutes, and then store at -80℃. Take out the tissue slide from the refrigerator, return to room temperature, wash the slide twice with PBS for 5 minutes, and transfer to 95℃ 1XpH6.0 sodium citrate for antigen repair for 30 minutes. Take out the staining jar, cool to room temperature, and then take out the slide. Transfer the slide to PBS and soak twice for 5 minutes each time. Prepare 5% BSA blocking solution with PBS, add blocking solution (100-400μL) to the tissue, and block at room temperature for 1 hour. Wipe off the excess blocking solution on the slide, add the prepared primary antibody (Anti-Rabbit Ki67 antibody; Anti-Rabbit monoclonal Olig2antibody; Rabbit-anti-Tuj1 / β-Tubulin III polyclonal antibody; Anti-CD31 Antibody; IBA1Guinea pigpAb; Mouse IgG2a-anti-GFAPmonoclonal antibody) to the tissue (100-400μL), and incubate overnight at 4℃ in a humidified chamber. The next day, wipe off the excess primary antibody, soak the slide in PBS 4 times, 5 minutes each time. Add the prepared secondary antibody to the tissue (100-400μL), and incubate for 1 hour in a humidified chamber at room temperature, away from light. Wipe off the excess secondary antibody (Goatanti-Rabbit IgG(H+L)-594; Goat anti-Rabbit IgG(H+L)-488; Goat anti-Mouse IgG(H+L)-488; Goat anti-Mouse IgG(H+L)-594; anti-Guinea pig IgG(H+L)-488; anti-Guineapig IgG(H+L)-647;), and soak the slide in PBS for 3 times, 5-10 minutes each time. DAPI with a final concentration of 0.1μg / mL can be added to the first PBS and incubated for 5 minutes; seal the slide and take pictures after the slide is dry.

[0099] The results of the identification of cultured medulloblastoma organoids are as follows Figure 3 to Figure 5 As shown. Figure 3As shown, the present invention uses HE staining for histological analysis to confirm that CBOs retain the characteristics of medulloblastoma; uses CD31 to mark the vasculature to prove that the organoids retain the vasculature of the parent tumor; uses Iba1 to mark microglia to prove that the organoids retain the immune cells of the parent tumor; uses Olig2 to mark oligodendrocytes to prove that the organoids retain the glial cell characteristics of the parent tumor; and uses Ki67 to mark proliferating cells to prove that the organoids retain the proliferative characteristics of the parent tumor. Figure 4 As shown, the present invention uses GFAP to mark astrocytes and β3-Tubulin to mark neurons, and the expression of both proves that the organoid retains the astrocyte and neuron cell types of the parent tumor. This result proves that the organoid has a high degree of similarity with the parent tumor.

Claims

1. Culture medium for culturing medulloblastoma organoids, It is characterized in that The organoids to be cultured are medulloblastoma organoids; the culture medium comprises the following components: basal culture medium, KOSR with a final concentration of 1%-10%, GlutaMax with a final concentration of 1X, AA with a final concentration of 1X, CDLC with a final concentration of 1%-3%, N2 with a final concentration of 1X, and B27 with a final concentration of 1X.

2. The culture medium according to claim 1, It is characterized in that The basic culture medium includes DMEM:F12 and Neurobasal; the volume ratio of DMEM:F12 to Neurobasal is 1-5:1-5.

3. The culture medium according to claim 1, It is characterized in that The culture medium further comprises Heparin with a final concentration of 3 to 8 μg / mL, T3 with a final concentration of 0.3 to 0.8 ng / mL, and BDNF with a final concentration of 10 to 20 ng / mL.

4. Methods for culturing medulloblastoma organoids without enzymatic digestion, It is characterized in that The steps include: (1) taking medulloblastoma tumor tissue, directly cutting it into pieces and then performing red blood cell lysis to obtain small tumor pieces; the medulloblastoma tumor tissue does not need to be subjected to enzyme digestion treatment; (2) culturing the small tumor mass obtained in step (1) using the culture medium of claim 1 to generate medulloblastoma organoids; (3) The medulloblastoma organoids obtained in step (2) are subcultured using the culture medium of claim 1 to obtain medulloblastoma organoids.

5. The method according to claim 4, It is characterized in that In step (2) and step (3), the culture conditions are: 37±5°C, 5±2% CO 2 , cultured on a horizontal shaker in a cell culture incubator at 90±5% relative humidity, with the rotation speed set to 80±5 rpm on the first day and 100±5 rpm in the later stage.

6. The method according to claim 4, It is characterized in that The medulloblastoma organoids are cryopreserved by the following method: the medulloblastoma organoids are incubated with a medulloblastoma organoid culture medium containing 8-15 μM Y-27632, and then cryopreserved with a cryopreservation solution; the cryopreservation solution consists of a medulloblastoma organoid culture medium, Y-27632 and DMSO; the content of Y-27632 in the cryopreservation solution is 8-15 μM, and the content of DMSO is 10%.

7. The method according to claim 4, It is characterized in that The medulloblastoma organoids are revived by the following method: after thawing the frozen medulloblastoma organoids, medulloblastoma organoid culture medium containing 8-15 μM Y-27632 and the freezing solution described in claim 6 are added for overnight static culture; on the next day, culture is carried out on a horizontal shaker using a culture medium without 8-15 μM Y-27632.

8. Medulloblastoma organoids cultured using the method according to any one of claims 4 to 7.

9. The medulloblastoma organoid according to claim 8, It is characterized in that The medulloblastoma organoids retain the neuroblastoma rosette or pseudorosette structure of the parental tumor; and / or retain the cell types of the parental tumor: β3-tubulin, GFAP, iba1, olig2 and CD31.

10. Use of the medulloblastoma organoid according to any one of claims 8 to 9 in the preparation of drugs for screening anti-medulloblastoma and / or in the construction of a medulloblastoma animal model.

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