Method for constructing and culturing neuroglioma organoid and complete culture medium of neuroglioma organoid

Abstract

The invention discloses a construction and culture method of a glioma organ and a complete culture medium thereof, a glioma tissue sample derived from a patient operation is subjected to tissue treatment processes such as physical cutting, enzymolysis digestion and red blood cell lysis to release a tumor cell cluster, and then the tumor cell cluster is resuspended in a carbon dioxide incubator by using the complete culture medium for shake culture. The tissue treatment process is simple, easy to operate and short in flow, extracellular matrixes such as matrigel do not need to be added in the culture process, the problem that standardization is difficult due to batch difference of the matrigel is solved, and the culture cost is reduced. The self-prepared complete culture medium is clear and simple in formula, and can maintain relatively high proliferation speed and organoid form of the neuroglioma organoid and long-term subculture. The glioma organoid constructed by the method has the advantages of high fitting degree, short culture period, low cost, easiness in standardization, suitability for high-throughput application and the like, and the modeling problem of the glioma organoid is well solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and specifically relates to the construction and culture method of glioma organoids and their complete culture medium. Technical Background

[0002] Organoids are three-dimensional cell cluster structures formed by in vitro culture of pluripotent stem cells or adult stem (progenitor) cells resident in adult tissues. Although organoids are not true human organs, they have the cell types and complex spatial morphology of the corresponding tissues and organs of stem cells, and can show the interactions and spatial position morphology between cells and between cells and their surrounding matrix. Moreover, they can simulate some functions and physiological responses of tissues and organs, and have a high similarity with the source tissues. At present, tumor organoids are the most widely used and clinically studied field in the field of organoids. Compared with traditional cancer cell line models and patient-derived tumor xenograft models (PDX), tumor organoids are a model with a short cycle, high efficiency, low cost, and can accurately reflect the pathophysiological state of the disease. They have great application potential in studying the occurrence and development of cancer, tumor heterogeneity, tumor gene-drug association, tumor target prediction, and drug sensitivity testing.

[0003] Glioma is a tumor originating from glial cells. It is a common primary intracranial malignant tumor, accounting for 27% of all primary central nervous system tumors and 80% of central nervous system malignant tumors. Every year, 5-8 people per 100,000 people suffer from glioma, and the 5-year mortality rate of glioma ranks third after pancreatic cancer and lung cancer. Glioma is one of the tumor types with a poor prognosis. The average survival of patients does not exceed one year, and it is difficult to perform R0 resection surgery. There is currently no effective drug to control glioma. Therefore, a new technical means for personalized medication of glioma is imperative, and the establishment and development of glioma organoids as an in vitro tumor model that can stably maintain the characteristics of tumor cells in vivo provides a new option for basic research and clinical precision treatment of glioma.

[0004] Glioma organoids are a three-dimensional cell culture system that mimics the microenvironment of tumors in the human body. Therefore, they are widely used to study the biological characteristics of gliomas, drug screening, and the development of treatment strategies. Although glioma organoids have been successfully constructed, there are some challenges in the existing construction methods: the traditional glioma organoid construction process generally involves enzymatic hydrolysis of tumor tissue to obtain single cells, which are then resuspended in low-growth factor matrix gel for 3D culture. However, the matrix gel is of animal origin, and there are some differences between batches, which may cause certain deviations in downstream drug sensitivity testing; the culture cycle of this method is generally 2 weeks to obtain organoids, but the growth rate of organoids varies greatly depending on the patient. The formation from single cells to organoids sometimes takes more than 1 month, and the timeliness cannot be guaranteed when guiding medication for precise treatment of patients; and the complete culture medium used in some glioma organoids is complex and costly, which makes it difficult to repeat in different laboratories. In 2021, a new method for constructing glioma organoids was reported. This method directly cultures shredded tissue fragments and obtains organoids from tissue fragments where tumor cells are concentrated. Organoids can be obtained in 2 weeks. This method avoids the use of matrix gel and the composition of the culture medium is relatively simple. However, this method has high requirements for the initial trimming of the tissue, and after one week of culture, it is necessary to remove tissue fragments where non-tumor cells are concentrated under a dissecting microscope. This process requires the operator to have rich experience and is prone to operational deviations. It is not easy to repeat and will cause the loss of sparse tissue samples of tumor cells.

[0005] Therefore, how to solve the problems of matrix gel batch differences, culture cycle inconsistency, complex complete culture medium components, and high requirements for tissue pruning in the culture of glioma organoids, and establish a simpler, easier-to-operate, shorter-cycle, and lower-cost glioma organoid construction has become a technical problem that needs to be urgently solved by technical personnel in this field. Summary of the invention

[0006] The first object of the present invention is to provide a method for constructing glioma organoids in response to the problems in the prior art.

[0007] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A method for constructing a glioma organoid, characterized in that it comprises the following steps:

[0009] In step 1, glioma tumor samples were obtained from surgical resection, placed in GDM medium to maintain cell activity and stability, and transported to the laboratory on ice;

[0010] Step 2: Pre-treat fresh glioma tissue to remove blood clots, necrotic tissue and other impurities, wash with ice-cold PBS three times, transfer the trimmed tissue to a clean culture dish, and cut it into 0.5-1mm on ice. 2 size;

[0011] Step 3: resuspend the chopped tissue in Advanced DMEM / F12 tissue digestion solution containing 1.5 mg / ml collagenase type 1 and 10 μM Y27632, place in a C02 incubator at 37°C, 5% C02, and 140 r / min for digestion, and use a pipette to fully blow every 10 minutes to observe the release of tumor cell clusters under a microscope;

[0012] Step 4: After the tumor cell clusters are fully released, add an appropriate amount of serum to terminate digestion, then centrifuge at 1000 r / min, 4°C and discard the supernatant;

[0013] Step 5: Add an appropriate amount of red blood cell lysis buffer, remove red blood cells, centrifuge at 1000 r / min, 4° C. and discard the supernatant to obtain tumor cell pellets.

[0014] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions:

[0015] As a preferred technical solution of the present invention: in step 1, the GDM medium is prepared by Hibernate-A, 1% GlutaMAX supplement, and 1% antibiotic antimycotic, Hibernate-A is selected as the basal medium, GlutaMAX supplement is selected as the glutamine required for cell growth, and antibiotic antimycotic is selected as an additive to prevent microbial contamination during cell culture.

[0016] The second object of the present invention is to provide a method for culturing glioma organoids in response to the problems in the prior art.

[0017] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0018] A method for culturing glioma organoids, characterized in that:

[0019] The following steps are involved:

[0020] S1, resuspended in 4 ml of complete glioma medium into an untreated 6-well plate, placed in a CO2 incubator at 37°C, 5% CO2, and shaken at 120 r / min;

[0021] S2, the glioma complete culture medium was replaced every 3 days, and the outgrowth of glioma organoids could be observed as soon as 1 day;

[0022] In step S1, the complete glioma culture medium includes: Advanced DMEM / F12 culture medium accounting for 50% of the culture medium, neurobasal culture medium accounting for 50% of the culture medium, 1×N2 additive, 1×B27 additive, 50 ng / mL bFGF, and 25 ng / mL EGF.

[0023] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions:

[0024] As a preferred technical solution of the present invention: the cultured glioma organoids are divided into 2 or 4 equal parts using ophthalmic scissors for passaging.

[0025] The third object of the present invention is to provide a complete culture medium for glioma organoids in response to the problems in the prior art.

[0026] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0027] The complete culture medium for glioma organoids is characterized by comprising: Advanced DMEM / F12 culture medium accounting for 50% of the culture medium, neurobasal culture medium accounting for 50% of the culture medium, 1×N2 additive, 1×B27 additive, 50 ng / mL bFGF, and 25 ng / mL EGF.

[0028] Compared with the prior art, the construction and culture method of the glioma organoid of the present invention and its complete culture medium provide an effective, stable and easy-to-operate method for culturing human glioma organoids in vitro, providing a reference for doctors to conduct high-throughput drug screening and clinical individualized treatment. Specifically, the method of the present invention does not need to use extracellular matrix materials such as matrigel: it avoids the differences in organoids caused by differences in matrix materials, and is more conducive to standardization; the suspension culture method is more conducive to large-scale culture: the operation is not affected by temperature, and is more conducive to high-throughput drug screening; the formation cycle of glioma organoids is short: the formation of organoids can be seen within 3 days using the method of the present invention, and mature passage can be achieved within 7 days, which greatly shortens the construction cycle of glioma organoids and saves precious time for patients to guide medication in vitro. The construction and culture method of the glioma organoid of the present invention and its complete culture medium improve the efficiency and accuracy of glioma organoid culture and are easy to promote and apply.

[0029] (1) The brain glioma organoids cultured in the culture medium of the present invention can be rapidly and long-term expanded in vitro, can be used for high-throughput screening, and are helpful for achieving large-scale replication and drug testing;

[0030] (2) Compared with the traditional organoid culture method, the culture method of the present invention does not require the use of matrix gel, thus avoiding the culture errors caused by unclear matrix gel composition and batch differences, improving repeatability, and greatly saving culture costs.

[0031] (3) The culture method of the present invention is simple to operate, less affected by human operation, and has stable culture results. It is practical and reliable and is suitable for application in standardized processes.

[0032] (4) The culture method of the present invention can efficiently and quickly culture organoids from glioma samples derived from patient tumor tissues in a short cycle, providing doctors with clinical individualized treatment drug sensitivity screening and providing a reference for personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 This is the growth morphology of the glioma organoids cultured in Example 1. Cases 1 and 2 shown in the figure are the organoid morphology at 3 days of culture, and Case 3 is the organoid morphology at 7 days of culture.

[0035] Figure 2 This is the growth morphology of the glioma organoids cultured using the method used in Example 2 after 14 days of continuous culture.

[0036] Figure 3 This is the growth morphology of the glioma organoids that were continuously subcultured using the culture medium of the present application in Example 3.

[0037] Figure 4 This is the growth morphology of the glioma organoids that were continuously subcultured using the culture medium of Scheme 1 in Example 4.

[0038] Figure 5 This is the growth morphology of the glioma organoids that were continuously subcultured using the culture medium in Scheme 2 in Example 5.

[0039] Figure 6 This is the growth morphology of the glioma organoids that were continuously subcultured using the culture medium of Scheme 3 in Example 6.

[0040] Figure 7 The results of HE staining of glioma organoids cultured according to the method used in this patent application. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0042] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0043] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods. The present application will be specifically described below through examples combined with the accompanying drawings.

[0044] Example 1: The method provided in the present invention is used to construct and culture glioma organoids.

[0045] like Figure 1 As shown, this embodiment provides a method and culture medium for culturing human glioma organoids, specifically comprising:

[0046] 1. Tissue processing:

[0047] (1) Glioma tumor samples were obtained from surgical resection, placed in GDM (composed of basal medium Hibernate-A, 1% GlutaMAX supplement, 1% antibiotic antimycotic), and transported to the laboratory on ice;

[0048] (2) Pre-treat fresh glioma tissue to remove blood clots, necrotic tissue and other impurities, wash with ice-cold PBS three times, transfer the trimmed tissue to a clean culture dish, and cut it into 0.5-1 mm on ice. 2 size;

[0049] (3) The minced tissue was resuspended in Advanced DMEM / F12 tissue digestion solution containing 1.5 mg / ml collagenase type 1 and 10 μM Y27632, and placed in a CO2 incubator at 37°C, 5% CO2, and 140 rpm for digestion. The tissue was fully blown every 10 minutes with a pipette, and the release of tumor cell clusters was observed under a microscope.

[0050] (4) After the tumor cell clusters are fully released, an appropriate amount of serum is added to terminate digestion, and then the mixture is centrifuged at 1000 r / min and 4°C and the supernatant is discarded;

[0051] (5) Add an appropriate amount of red blood cell lysis buffer, remove red blood cells, centrifuge at 1000 r / min, 4°C and discard the supernatant to obtain tumor cell pellets.

[0052] 2. Organoid Culture:

[0053] (1) Resuspend the cells in 4 ml of complete glioma medium into an untreated 6-well plate and place in a CO2 incubator at 37°C, 5% CO2, and 120 rpm shaking.

[0054] (2) Glioma complete culture medium was replaced every 3 days, and the outgrowth of glioma organoids could be observed as soon as 1 day;

[0055] (3) When the glioma organoids grow larger, they can be divided into two or four equal parts using ophthalmic scissors for passaging.

[0056] 3. Complete culture medium formula:

[0057] Specific ingredients include: Advanced DMEM / F12 medium (50%), neurobasal medium (50%), N2 additive (1×), B27 additive (1×), bFGF (50 ng / mL), and EGF (25 ng / mL).

[0058] In the present invention, the GDM medium is a nutrient medium specifically used for cell culture.

[0059] PBS refers to phosphate buffered saline.

[0060] bFGF refers to fibroblast growth factor.

[0061] EGF, which stands for epidermal growth factor.

[0062] Example 2: Cultivation of glioma organoids using the matrix gel embedding method.

[0063] like Figure 2 As shown, this embodiment provides a method for culturing human glioma organoids by matrix gel embedding. The tissue processing process and the culture medium used are the same as those in Example 1. The specific culture method is as follows:

[0064] (1) Dissolve the matrix gel on ice in advance, resuspend the dissociated tumor cell clusters in 200 μL of matrix gel, and evenly spread them in an untreated 6-well plate, with each drop being about 20 μL;

[0065] (2) Invert at room temperature for 5 min, then transfer to a CO2 incubator and invert for 20 min. After the matrix gel solidifies, add complete culture medium and incubate in a CO2 incubator at 37°C, 5% CO2.

[0066] (3) The glioma complete culture medium was replaced every 3 days, and the growth of glioma organoids was observed every day.

[0067] Example 3: The culture medium of the present application is used to continuously subculture glioma organoids.

[0068] like Figure 3 As shown, this example uses the construction method and culture medium in Example 1 to continuously subculture the glioma organoids and observe the growth status of the organoids. The specific method is as follows:

[0069] (1) When the organoid grows to a diameter greater than 1 mm, use ophthalmic scissors to divide the glioma organoid into four equal parts. This operation can be performed under a stereomicroscope.

[0070] (2) The cells were resuspended in complete medium and plated in an untreated 6-well plate at a subculture ratio of 1:4. The cells were cultured in a CO2 incubator at 37°C, 5% CO2, and 120 rpm shaking.

[0071] (3) The glioma complete culture medium was replaced every 3 days, and the growth of glioma organoids was observed every day.

[0072] The present invention discloses a novel method for constructing and culturing glioma organoids, and a complete culture medium for glioma organoids. The glioma tissue samples obtained from patient surgery are subjected to tissue processing processes such as physical shearing, enzymatic digestion, and red blood cell lysis to release tumor cell clusters, which are then resuspended in a complete culture medium and placed in a carbon dioxide incubator for shaking culture. The tissue processing process is simple and easy to operate, with a short process. The culture process does not require the addition of extracellular matrices such as matrix gel, which avoids the problem of difficulty in standardization caused by differences in matrix gel batches and reduces the cost of culture. The formula of the self-configured complete culture medium is clear and simple, and can maintain the faster proliferation rate and organoid morphology of glioma organoids and long-term subculture. The glioma organoids constructed by this method have the advantages of high fitting, short culture cycle, low cost, easy standardization, and suitability for high-throughput applications, which well solves the modeling problem of glioma organoids.

[0073] Example 4, Scheme 1: Continuous subculture of glioma organoids in culture medium.

[0074] like Figure 4 As shown, this embodiment provides a glioma organoid culture medium (Scheme 1), the specific components are: Advanced DMEM / F12 medium (50%), neurobasal medium (50%), N2 additive (1×), B27 additive (1×), EGF (25 ng / mL). The tissue processing and culture methods are the same as in Example 1, and the glioma organoids are continuously subcultured.

[0075] Example 5, Scheme 2: Continuous subculture of glioma organoids using culture medium.

[0076] like Figure 5 As shown, this embodiment provides a glioma organoid culture medium (Scheme 2), the specific ingredients are: Advanced DMEM / F12 medium (50%), neurobasal medium (50%), N2 additive (1×), B27 additive (1×), EGF (25ng / mL), GlutaMax (1×), N-acetylcysteine ​​(0.5mM), Y27632 (20μM), Nicotinamide (5mM), MEM-NEAAs (1×). The tissue processing and culture methods are the same as in Example 1, and the glioma organoids are continuously subcultured.

[0077] Example 6, Scheme 3 medium for continuous subculturing of glioma organoids.

[0078] like Figure 6 As shown, this embodiment provides a glioma organoid culture medium (Scheme 3), the specific ingredients are: Advanced DMEM / F12 medium (50%), neurobasal medium (50%), N2 additive (1×), B27 additive (1×), GlutaMax (1×), N-acetylcysteine ​​(0.5mM), MEM-NEAAs (1×), 2-mercaptoethanol (0.1%), human insulin (375ng / ml). The tissue processing and culture methods are the same as in Example 1, and the glioma organoids are continuously subcultured.

[0079] Example 7. Morphological identification of tumor tissues and glioma organoids cultured in Example 1.

[0080] like Figure 7 As shown, in this example, the human glioma organoids and primary tumor tissues obtained in Example 1 were paraffin-embedded to prepare sections. The embedded organoids and tissues were sectioned and then HE-stained for observation.

[0081] Finally, it should be noted that the terms "comprises", "includes" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or device. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for constructing glioma organoids, characterized in that: The following steps are involved: Step 1: Obtain glioma tumor samples from surgical resection to obtain glioma tissue, which is then placed in GDM medium to maintain cell activity and stability and transported on ice; Step 2: Pre-treat the glioma tissue, remove impurities, wash with ice-cold PBS, and cut the pre-treated glioma tissue into 0.5-1 mm pieces on ice. 2 size; Step 3: resuspend the minced glioma tissue in Advanced DMEM / F12 tissue digestion solution containing 1.5 mg / ml collagenase type 1 and 10 μM Y27632, and place it in a CO2 incubator at 37°C, 5% CO2, and 140 r / min for digestion. Use a pipette to fully blow every 10 minutes until the tumor cell clusters are fully released. Step 4: After the tumor cell clusters are fully released, serum is added to terminate digestion, and then centrifuged at 1000 r / min, 4°C and the supernatant is discarded; Step 5: Add red blood cell lysis buffer, remove red blood cells, centrifuge at 1000 r / min, 4° C. and discard the supernatant to obtain tumor cell pellets.

2. The method for constructing glioma organoids according to claim 1, characterized in that: In step 1, the GDM medium is prepared from Hibernate-A, 1% GlutaMAX supplement, and 1% antibiotic antimycotic. Hibernate-A is selected as the basal medium, GlutaMAX supplement is selected as glutamine required for cell growth, and antibiotic antimycotic is selected as an additive to prevent microbial contamination during cell culture.

3. A method for culturing glioma organoids constructed using the method of claim 1 or 2, characterized in that: The following steps are involved: S1, resuspend the glioma tumor cell cluster with 4 ml of glioma complete medium, add the resuspended cell suspension to an untreated 6-well plate, and place the 6-well plate in a CO2 incubator at 37°C, 5% CO2, and 120 r / min shaking culture; S2, replace glioma complete medium every 3 days to maintain cell growth and proliferation until glioma organoids are generated; In step S1, the complete glioma culture medium includes: Advanced DMEM / F12 culture medium accounting for 50% of the culture medium, neurobasal culture medium accounting for 50% of the culture medium, 1×N2 additive, 1×B27 additive, 50 ng / mL bFGF, and 25 ng / mL EGF.

4. The method for culturing glioma organoids according to claim 3, wherein: Cultured glioma organoids were divided into 2 or 4 equal parts using ophthalmic scissors for passage.

5. A complete culture medium for glioma organoids, characterized in that: The medium includes Advanced DMEM / F12 medium accounting for 50% of the medium, neurobasal medium accounting for 50% of the medium, 1×N2 supplement, 1×B27 supplement, 50 ng / mL of bFGF, and 25 ng / mL of EGF.

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