Methods for constructing brain glioma organoids

Abstract

The present invention relates to the technical field of organoids, and discloses a method for constructing brain glioma organoids. The method provided by the present invention can realize the construction and expansion of glioma organoids under static conditions by utilizing a culture device with a specific structure, avoiding the use of additional equipment such as a shaker, reducing the requirements for an incubator, and also reducing the risk of microbial infection during the culture process. In addition, the method provided by the present invention can better simulate the human immune microenvironment, and improve the experimental reference value of the glioma organoids obtained by the method.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and in particular to a method for constructing brain glioma organoids. Background Art

[0002] Organoids are three-dimensional cell cultures that have highly similar histological features to corresponding human organs and can reproduce some of the physiological functions of organs in vitro. The emergence of organoids provides researchers with a new in vitro model with unique advantages, which can replicate the complex spatial morphology of differentiated tissues and can show the interactions between cells and cells and cells and matrix. Organoids remove the confounding variables that may be introduced by animal models, but provide a higher degree of complexity compared to homogenized 2D cell cultures. By combining a high level of physiological relevance with the convenience of in vitro operations, in many cases, organoids have the potential to assist or replace in vitro experiments using primary cells or immortalized cell lines and animals. In addition, organoids have a high degree of genetic stability in culture and maintain the genotype and phenotype of the source tissue. Therefore, organoids have broad application prospects in many fields such as stem cells and development, regenerative medicine, disease research, drug development and precision medicine.

[0003] Glioblastoma is the most common primary intracranial tumor. In order to better study the pathogenic mechanism and treatment of glioma, scientists have constructed glioma organoids. For example, Jacob et al. introduced the use of mechanical fragmentation to cut glioma tissue into 1 mm 3 The fragments are then placed in culture medium and cultured on a horizontal shaker. In this method, the shaker is set up to provide sufficient air exchange for the organoids during the culture process. However, the incubators usually used to culture organoids adopt 5% CO2 culture conditions. Long-term exposure to CO2 can easily cause damage to the shaker. Moreover, the shaker will take up a lot of space in the incubator. On the other hand, the need for this additional equipment has certain requirements for both the incubator and the shaker, and it also increases the risk of bacterial contamination of the culture. In addition, this method also has high requirements for the volume and quality of glioma samples, and the success rate of culture needs to be further improved. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems in the prior art of glioma organoid construction methods that require additional equipment such as a shaker to be placed in the incubator, have high requirements for samples, and have an insufficient culture success rate. A method for constructing brain glioma organoids is provided, which can realize the construction and expansion of glioma organoids under static conditions and has a high culture success rate.

[0005] In order to achieve the above-mentioned purpose, the present invention provides a method for constructing glioma organoids on one hand, characterized in that the method comprises placing a glioma sample coated with matrix gel in an air-liquid interface culture device for culture, wherein the air-liquid interface culture device is filled with a culture medium, and the amount of the culture medium is such that no more than 50% of the volume of the sample is immersed therein.

[0006] The second aspect of the present invention provides a culture medium for constructing brain glioma organoids in the method described in the first aspect.

[0007] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0008] (1) The method provided by the present invention uses an air-liquid interface culture device to culture glioma organoids. The air exchange of the glioma organoids is ensured by the air-liquid interface culture device, thereby eliminating the need for additional equipment such as a shaker, reducing the requirements for an incubator, and also reducing the risk of microbial infection during the culture process.

[0009] (2) The method provided by the present invention first coats the glioma tissue with matrix gel and then cultures it in a culture medium. Compared with the culture method in the prior art of directly placing the organoids in a culture medium, this method can better simulate the human immune microenvironment and improve the experimental reference value of the glioma organoids prepared by the method provided by the present invention.

[0010] (3) In order to obtain a higher culture success rate, the glioma culture methods in the prior art usually require a larger sample volume and usually require high-quality tumor parenchyma, which greatly limits the source of samples. The method provided by the present invention does not require a large sample volume and does not require high sample quality, but the method can construct glioma organoids with a higher success rate.

[0011] (4) The method provided by the present invention not only reduces the amount of glioma samples used, but also greatly saves the amount of culture medium used, which is very helpful in controlling experimental costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the structure of the gas-liquid interface culture device used in the method provided by the present invention.

[0013] Figure 2 This is a morphological image of the brain glioma organoid obtained in Example 1 (the image legend is 200 μm).

[0014] Figure 3 Immunofluorescence staining of the glioma organoids obtained in Example 1.

[0015] Description of Reference Numerals

[0016] Figure 1 In the figure, 1 is the inner culture dish and 2 is the outer culture dish. DETAILED DESCRIPTION

[0017] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0018] During the construction and passage of glioma organoids, it is necessary to maintain a certain air content in the culture system so that the glioma organoids can be fully in contact with the air, thereby promoting their growth. Therefore, it is usually necessary to place a shaker in the incubator when glioma organoids are cultured, and the amount of air dissolved in the culture medium is ensured by shaking culture. The inventors of the present invention cleverly discovered in the process of research that by rationally designing the structure of the culture dish, the contact area of ​​the glioma organoids with the air during the culture process can be effectively increased, so that they can be cultured under static conditions, avoiding the use of a shaker. This can not only reduce the requirements for the incubator (for example, in the traditional method, an incubator with a large enough volume must be used to accommodate the shaker, and the remaining volume of the incubator after the shaker is placed also needs to be large enough to avoid the incubator being too crowded and causing air to stagnate, which is not conducive to the growth of glioma organoids), but also reduce the risk of microbial infection caused by the introduction of additional equipment.

[0019] After further research, the inventors designed a gas-liquid interface culture device suitable for culturing glioma organoids under static conditions ( Figure 1 The inventors also found that the use of the device in combination with a culture medium containing specific components for glioma organoid construction can effectively improve the success rate and quality of glioma organoid construction.

[0020] Based on the above findings, the present invention provides a method for constructing glioma organoids, which comprises placing a glioma sample coated with matrix gel in an air-liquid interface culture device for culture, wherein the air-liquid interface culture device is filled with a culture medium, and the amount of the culture medium is such that no more than 50% by volume of the sample coated with matrix gel is immersed therein.

[0021] Any matrix glue that can be used for organoid culture or 3D cell culture can be applied to the present invention. According to a preferred embodiment of the present invention, the pH of the matrix glue is not less than 7.3 (preferably 7.3-7.4). The present invention can directly use a matrix glue that meets the aforementioned pH conditions, or it can condition the pH value of the matrix glue before use so that it meets the aforementioned requirements. In addition, the present invention has no particular restrictions on the source of the matrix glue, which can be a related product prepared by itself, or a related product directly obtained by commercial purchase or customization (for example, it can be purchased from Corning Company). basement membrane and other matrix gel products).

[0022] Preferably, the matrix gel is diluted with culture medium before use, and preferably the volume ratio of the culture medium to the matrix gel is 10-50:1.

[0023] More preferably, the volume ratio of the matrix gel used to coat the glioma sample to the glioma sample is 2-10:1.

[0024] Compared with the prior art, the method provided by the present invention can effectively reduce the volume requirement for glioma samples, realize the construction and culture of organoids using glioma samples of moderate or smaller volumes, reduce the requirements for raw material samples, and improve the universality of glioma organoid construction. According to some preferred embodiments of the present invention, the size of the brain glioma sample does not exceed 2cm×2cm×2cm. Preferably, it is 1-1.5cm×1-1.5cm×1-1.5cm.

[0025] According to a preferred embodiment of the present invention, the culture medium comprises a basal culture medium and additives, the basal culture medium is selected from at least one of DMEM / F-12, Neurobasal and Hibernate, and the additives include at least one of B-27 additive, N2 additive, basic fibroblast growth factor (FGF-2), vitamin A, vitamin C, insulin and β-mercaptoethanol.

[0026] Preferably, the additive further contains at least one of antibiotics, L-glutamine, non-essential amino acids, 4-hydroxyethylpiperazineethanesulfonic acid, and nicotinamide.

[0027] Any antibiotic suitable for use in organoid culture can be applied to the present invention. Preferably, the antibiotic is selected from at least one of penicillin, streptomycin and amphotericin B.

[0028] More preferably, the non-essential amino acid is selected from at least one of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine. In the present invention, there is no particular restriction on the source of the non-essential amino acid, which can be obtained by self-preparation or directly obtained from commercial or customized related products (such as commercially available NEAA composite additives, etc.).

[0029] According to a preferred embodiment of the present invention, in the culture medium, the additives are used in an amount such that their contents in the culture medium are: antibiotics 1-2 volume % (if a composite antibiotic is used, the content here is the total content of each antibiotic), L-glutamine 1-2 volume %, non-essential amino acids 1-2 volume % (if multiple non-essential amino acids are used, the content here is the total content of each non-essential amino acid), 4-hydroxyethylpiperazineethanesulfonic acid 1-2 volume %, nicotinamide 1-2 volume %, B-27 additive 2-4 volume %, N2 additive 1-4 volume %, basic fibroblast growth factor 2-10 nM, vitamin A 1-2 volume %, vitamin C 1-2 volume %, insulin 10-50 μg / mL, and β-mercaptoethanol 0.1-0.2 volume %.

[0030] Preferably, the antibiotics are used in an amount such that their final concentration in the culture medium is 100-200 U / mL of penicillin and 0.1-0.2 mg / mL of streptomycin, the L-glutamine is used in an amount such that its final concentration in the culture medium is 2-4 mM, the non-essential amino acids are used in an amount such that their final concentration in the culture medium is 100-200 μM (it should be noted that if multiple non-essential amino acids are used, the final concentration here refers to the final concentration of each non-essential amino acid alone), the 4-hydroxyethylpiperazineethanesulfonic acid is used in an amount such that its final concentration in the culture medium is 100-200 μM. The final concentration of the nicotinamide in the culture medium is 10-20 mM, the amount of nicotinamide used is such that its final concentration in the culture medium is 4-8 mM, the amount of the B-27 additive used is such that its final concentration in the culture medium is 0.5-5×, the amount of the N-2 additive used is such that its final concentration in the culture medium is 0.5-5×, the amount of vitamin A used is such that its final concentration in the culture medium is 200-400 μM, the amount of vitamin C used is such that its final concentration in the culture medium is 100-200 μM, and the amount of β-mercaptoethanol used is such that its final concentration in the culture medium is 50-100 μM.

[0031] More preferably, the amount of the non-essential amino acids is such that in the culture medium: the final concentration of glycine is 100-200 μM, the final concentration of alanine is 100-200 μM, the final concentration of asparagine is 100-200 μM, the final concentration of aspartic acid is 100-200 μM, the final concentration of glutamate is 100-200 μM, the final concentration of proline is 100-200 μM, and the final concentration of serine is 100-200 μM.

[0032] refer to Figure 1 According to a preferred embodiment of the present invention, the gas-liquid interface culture device comprises an outer culture dish and an inner culture dish, and the diameter of the outer culture dish is 1.5-3 times the diameter of the inner culture dish. In the gas-liquid interface culture device, the inner culture dish is placed in the outer culture dish. When glioma organoids are cultured, the matrix gel coated with the brain glioma sample (glioma tissue or glioma organoids) is placed in the inner culture dish, and the culture medium is added to the outer culture dish (the culture medium can penetrate into the inner culture dish through the holes or slits set at the bottom of the inner culture dish, thereby contacting the glioma organoids).

[0033] Preferably, the bottom of the inner culture dish contains holes or slits, so that the culture medium can enter the inner culture dish through the holes or slits and contact the matrix gel coated with the glioma sample. The size of the pore size or slit is sufficient to allow the culture medium to pass through, so that the components contained therein can fully contact the glioma sample. Preferably, the diameter of the hole (or the width of the slit) is 0.2-0.8 μm.

[0034] According to a preferred embodiment of the present invention, the bottom of the inner culture dish is made of a semipermeable membrane, and the semipermeable membrane contains holes of the size as described above for the culture medium to enter the inner culture dish. The present invention has no particular restrictions on the specific selection of the semipermeable membrane, as long as it can be suitable for glioma culture. Preferably, the culture medium and its components (such as various small molecule additives, etc.) and the metabolites secreted and expressed by the glioma organoids during the culture process can all pass through the semipermeable membrane.

[0035] The present invention has no particular restrictions on the size of the gas-liquid interface culture device, as long as it can meet the needs of glioma organoid culture. Preferably, the ratio of the diameter of the outer culture dish to the inner culture dish is 1.2-5. According to a preferred embodiment of the present invention, the diameter of the outer culture dish is 60-100mm, and the diameter of the inner culture dish is 30-50mm. By adopting a culture device of the above size, the volume of glioma tissue used to culture glioma organoids can be effectively controlled, thereby achieving the construction of glioma organoids with a moderate volume of tumor samples, and the quality requirements for glioma tissue used to construct organoids can be appropriately reduced (the prior art methods usually require the use of high-quality tumor parenchyma parts to construct glioma organoids with a higher success rate, while the method of the present invention only requires the use of glioma samples of moderate quality).

[0036] The gas-liquid interface culture device provided by the present invention can make the glioma organoids fully contact with the air, without the need to use additional equipment or means to increase the dissolved oxygen content in the culture medium. Therefore, it is preferred that the gas-liquid interface culture device does not include a shaker or oscillator.

[0037] According to a preferred embodiment of the present invention, the method further comprises a pretreatment step, and the pretreatment step preferably comprises pretreatment of the air-liquid interface culture device and pretreatment of the glioma sample.

[0038] According to a preferred embodiment of the present invention, the pretreatment of the air-liquid interface culture device includes laying a matrix glue layer in the inner culture dish before placing the glioma sample in the inner culture dish.

[0039] Preferably, the thickness of the matrix gel layer does not exceed 50% of the height of the inner culture dish, preferably does not exceed 3 mm. The function of the matrix gel layer is to provide a "platform" for the glioma tissue coated with matrix gel added later, so that it can have a certain height from the bottom of the culture dish, so that part of the glioma tissue coated with matrix gel can be exposed to the surface of the culture medium and fully contact with the air.

[0040] According to a preferred embodiment of the present invention, the pretreatment of the glioma sample includes washing and crushing the glioma sample.

[0041] Preferably, the cleaning comprises the steps of performing a first cleaning on the glioma sample before pulverization and performing a second cleaning on the pulverized glioma sample after pulverization.

[0042] Preferably, the (total) content of Mg and Ca in the cleaning reagent does not exceed 0.1 g / L.

[0043] According to a preferred embodiment of the present invention, the culture conditions include: a temperature of 36-38°C, and the culture medium is replaced when the evaporation of the culture medium reaches more than 50% by volume of the added amount, preferably once every 4-7 days.

[0044] The second aspect of the present invention provides a culture medium for constructing brain glioma organoids in the method as described above.

[0045] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to exemplarily explain and illustrate the content of the present invention, and are not used to limit the present invention.

[0046] The DPBS++ buffer used in the following examples is Mg 2+ and Ca 2+ The total content of PBS buffer is about 0.1 g / L. Unless otherwise specified, the reagents and materials used are commercial products purchased from regular chemical or biological reagent / material suppliers, and the reagents are all analytical grade.

[0047] Preparation Example 1

[0048] (I) Culture device

[0049] refer to Figure 1 The schematic diagram of the structure in the figure shows a gas-liquid interface culture device for culturing glioma organoids. The inner culture dish has a diameter of 30 mm and a height of 13 mm. The bottom of the inner culture dish is made of a semipermeable membrane made of hydrophilic polytetrafluoroethylene with holes of 0.4 μm in diameter. The outer culture dish has a diameter of 60 mm and a height of 15 mm.

[0050] (II) Culture medium

[0051] According to the formula in Table 1, DMEM / F-12 medium was used as the basic medium to prepare the medium for glioma organoid construction. Among them, B-27 additive was purchased from Thermo Fisher with a catalog number of 12587010, N-2 additive was purchased from Thermo Fisher with a catalog number of 17502001, vitamin A was purchased from Selleck with a catalog number of S1653, vitamin C was purchased from Selleck with a catalog number of S3114, β-mercaptoethanol was purchased from Thermo Fisher with a catalog number of 21985023, penicillin-streptomycin was purchased from Thermo Fisher with a catalog number of 15140163, L-glutamine was purchased from Thermo Fisher with a catalog number of 35050079, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) was purchased from Thermo Fisher with a catalog number of 15630130, and nicotinamide was purchased from MCE with a catalog number of HY-B0150. The non-essential amino acids were purchased from Thermo Fisher with a mixed amino acid supplement with product number 11140050, which contained glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine.

[0052] Table 1

[0053]

[0054] Example 1

[0055] The following method was used to construct brain glioma organoids. The glioma samples used were derived from tumor tissue removed from brain glioma patients after surgery, and informed consent was obtained from the relevant personnel.

[0056] First washing: The glioma tissue was washed three times with pre-cooled (4°C) DPBS++.

[0057] Sample crushing: The cleaned glioma tissue was transferred to a 10 cm sterile culture dish and cut into pieces with a particle size of about 0.5-1 mm using a sterile scalpel.

[0058] Second cleaning: The crushed glioma samples were repeatedly washed three times with room temperature DPBS++ buffer.

[0059] Red blood cell lysis: Add 10 mL of red blood cell lysis buffer (purchased from Thermo Fisher, catalog number 00433357) to the crushed glioma sample obtained after the second wash, and shake on a shaker at 10 rpm for 10 min at room temperature. After the lysis is completed, wash three times with room temperature DMEM / F12 medium to obtain the glioma tissue to be cultured.

[0060] Organoid plate: Take the air-liquid interface culture device obtained in Preparation Example 1, add 1 mL of Matrigel ( The mixture was then placed in a 37°C incubator for 30 min to allow the matrix gel to solidify and form a matrix gel layer with a thickness of about 1 mm.

[0061] Take an appropriate amount of glioma tissue to be cultured and mix it with 1 mL of matrix gel (the volume ratio of glioma tissue to matrix gel is about 1:5), then add the mixed gel solution to the inner culture dish, place it in a 37°C incubator and let it stand for 30 minutes to form a glioma tissue layer on the matrix gel layer. Then add 1 mL of the culture medium prepared in Preparation Example 1 to the outer culture dish (the amount of culture medium added is such that about 50% of the glioma tissue layer is immersed in the culture medium).

[0062] Organoid culture and passaging: Place the gas-liquid interface culture device in a 37°C incubator (5% CO2), observe the evaporation of the culture medium every day, and observe the culture of glioma organoids under a microscope. When the evaporation of the culture medium reaches 50%, replace it with fresh culture medium (1 mL). When the organoid grows to a diameter of 1-2 mm, pass the glioma organoid.

[0063] Figure 2 The microscopic picture of the brain glioma organoids cultured by the above method is shown. As can be seen from the figure, the glioma organoids obtained by the above method are evenly distributed in the inner culture dish, with a diameter of about 1 mm, smooth edges and a dense core.

[0064] A total of 50 glioma samples from patients with brain glioma were used to culture organoids according to the above method, and glioma organoids were successfully obtained (the morphological characteristics of glioma organoids obtained by culturing glioma samples were similar to those of Figure 2 The success rate of culture was 100% (50 / 50).

[0065] Immunofluorescence staining of the obtained glioma organoids revealed that these organoids retained the representative marker of brain glioma: astrocytes. The presence of astrocytes was determined by detecting glial fibrillary acidic protein (GFAP), a marker of astrocyte activation. Figure 3 As shown, the blue fluorescence in the figure is DAPI (representing the cell nucleus) and the green fluorescence is GFAP (representing astrocytes).

[0066] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for constructing brain glioma organoids, characterized in that: The method comprises placing a brain glioma sample coated with matrix gel in an air-liquid interface culture device for culture, wherein the air-liquid interface culture device is filled with a culture medium, and the amount of the culture medium is such that no more than 50% of the volume of the sample coated with matrix gel is immersed in the culture medium; The culture medium comprises a basal culture medium and additives, wherein the basal culture medium is selected from at least one of DMEM / F-12, Neurobasal and Hibernate, and the additives are used in an amount such that the final concentrations in the culture medium are: penicillin 100-200 U / mL, streptomycin 0.1-0.2 mg / mL, L-glutamine 2-4 mM, non-essential amino acids 100-200 μM, 4-hydroxyethylpiperazineethanesulfonic acid 10-20 μM, nicotinamide 4-8 μM, B-27 additive 0.5-5×, N-2 additive 0.5-5×, basic fibroblast growth factor 2-10 nM, vitamin A 200-400 μM, vitamin C 100-200 μM, insulin 10-50 μg / mL, and β-mercaptoethanol 50-100 μM; The gas-liquid interface culture device comprises an outer culture dish and an inner culture dish, wherein the diameter of the outer culture dish is 1.5-3 times the diameter of the inner culture dish; The bottom of the inner culture dish contains holes or slits; The gas-liquid interface culture device does not include a shaker or oscillator; The method further comprises laying a matrix gel layer in the inner culture dish before placing the glioma sample in the inner culture dish.

2. The method according to claim 1, wherein: The pH of the matrix gel is not lower than 7.

4.

3. The method according to claim 1, wherein: The matrix gel is diluted with culture medium before use.

4. The method according to claim 3, wherein: The volume ratio of culture medium to Matrigel used for dilution was 10-50:

1.

5. The method according to claim 1, wherein: The volume ratio of the matrix gel used to coat the glioma sample to the glioma sample is 2-10:

1.

6. The method according to claim 1, wherein: The size of the brain glioma sample does not exceed 2 cm×2 cm×2 cm.

7. The method according to claim 6, wherein: The size of the brain glioma sample is 1-1.5 cm×1-1.5 cm×1-1.5 cm.

8. The method according to claim 1, wherein: The non-essential amino acid is selected from at least one of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine.

9. The method according to claim 1, wherein: In the culture medium, the additives are used in such amounts that the content thereof in the culture medium is: 1-2% by volume of antibiotics, 1-2% by volume of L-glutamine, 1-2% by volume of non-essential amino acids, 1-2% by volume of 4-hydroxyethylpiperazineethanesulfonic acid, 1-2% by volume of nicotinamide, 2-4% by volume of B-27 additive, 1-4% by volume of N-2 additive, 1-2% by volume of vitamin A, 1-2% by volume of vitamin C, and 0.1-0.2% by volume of β-mercaptoethanol.

10. The method according to claim 1, wherein: The diameter of the holes is 0.2-0.8 μm.

11. The method according to claim 1, wherein: The diameter of the outer culture dish is 60-100 mm, and the diameter of the inner culture dish is 30-50 mm.

12. The method according to claim 1, wherein: The method further comprises a pre-treatment step.

13. The method according to claim 12, wherein the pretreatment step comprises pretreatment of a glioma sample.

14. The method according to claim 13, wherein: The pretreatment of the glioma sample includes cleaning and crushing the glioma sample.

15. The method according to claim 14, wherein: The cleaning comprises the steps of performing a first cleaning on the glioma sample before pulverization and performing a second cleaning on the pulverized glioma sample after pulverization; And / or, the content of Mg and Ca in the reagent used for cleaning does not exceed 0.1 g / L.

16. The method according to claim 1, wherein: The thickness of the matrix gel layer does not exceed 50% of the height of the inner culture dish.

17. The method according to claim 16, wherein: The thickness of the matrix glue layer does not exceed 3 mm.

18. The method according to claim 1, wherein: The culture conditions include: a temperature of 36-38° C., and replacing the culture medium when the evaporation amount of the culture medium reaches more than 50% by volume of the added amount.

19. The method according to claim 18, wherein: The culture medium was changed every 4-7 days.

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