Matrigel for brain organoid differentiation and its preparation method and application

Abstract

The present invention discloses a Matrigel for brain organoid differentiation, its preparation method, and application. The Matrigel comprises, by volume, 20%-40% GFR, 50%-70% type I collagen, 0.5%-2% β-mercaptoethanol, and 8%-9.5% Neurobasal+B27. The Matrigel provided by the present invention can improve the differentiation efficiency of brain organoids, enhance the stability of the experimental system, and reduce costs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a matrix glue for brain organoid differentiation, and its preparation method and application. The provided matrix glue can improve the differentiation efficiency of brain organoids, improve the stability of the experimental system, and reduce actual costs. Background Art

[0002] In research on stem cell differentiation into brain-like tissue (also referred to herein as brain organoids), specific types of matrix gels are often used to provide a three-dimensional environment supporting cell growth and differentiation. These matrix gels mimic the properties of the extracellular matrix in vivo and are crucial for maintaining cell adhesion, morphology, and function, as well as promoting cell proliferation and differentiation. Currently used hydrogel matrices or matrix gels primarily include mixed extracellular matrices derived from animal tumor cells or tissues (such as Matrigel and Cultrex® BME), synthetic polypeptide hydrogels (such as PuraMatrix), gelatin, and synthetic hydrogels (such as polyethylene glycol and polyacrylamide) (see, for example, WO2024233916A3, WO2024023668A1, CN119630778A, and CN115612657A). These hydrogels or matrix gels often suffer from significant batch-to-batch variability, unclear composition, and / or poor bioactivity.

[0003] Therefore, there is an urgent need in the art for an improved Matrigel for brain organoid differentiation. Summary of the Invention

[0004] In order to solve one or more problems existing in the prior art, one aspect of the present invention provides a matrix gel, which may comprise, or consist of, 20%-40% GFR, 50%-70% type I collagen, 0.5%-2% β-mercaptoethanol, and 8%-9.5% Neurobasal+B27 in terms of volume percentage;

[0005] The Neurobasal+B27 refers to a Neurobasal culture medium supplemented with 2% by volume of B27.

[0006] In some embodiments, the matrix gel may comprise, or consist of, 25%-35% GFR, 55%-65% type I collagen, 1%-1.5% β-mercaptoethanol, and 8.5%-9% Neurobasal+B27 by volume.

[0007] In some embodiments, the matrix gel may comprise, or consist of, 30% GFR, 60% type I collagen, 1% β-mercaptoethanol, and 9% Neurobasal+B27 by volume.

[0008] Another aspect of the present invention provides a method for preparing matrigel, which comprises the following steps:

[0009] S1: Mix GFR and type I collagen in proportion under sterile conditions;

[0010] S2: adding β-mercaptoethanol to the product obtained in step S1 and stirring until completely dissolved; and

[0011] S3: Add Neurobasal+B27 to the product obtained in step S2 and mix thoroughly to obtain the matrix gel.

[0012] In another aspect, the present invention provides the use of the above-mentioned matrix gel in inducing stem cells to differentiate into brain organoids.

[0013] In some embodiments, the stem cells are induced pluripotent stem cells.

[0014] In yet another aspect, the present invention provides a method for inducing stem cell differentiation into brain organoids, comprising embedding embryoid bodies formed from stem cells in the aforementioned Matrigel and culturing them. Based on this method, the present invention also provides brain organoids obtained thereby, as well as applications of the brain organoids in developmental biology research, disease modeling, and drug screening.

[0015] The Matrigel provided by the present invention offers multiple advantages in brain organoid differentiation, better simulating the in vivo microenvironment and promoting cell growth, differentiation, and functional maturation. Specifically, the Matrigel provided by the present invention combines multiple specific components (such as GFR Matrigel, type I collagen, and β-mercaptoethanol), mimicking the complex structure and function of the in vivo extracellular matrix. It effectively provides the mechanical support and biochemical signals required for cell growth, promoting cell adhesion, migration, and differentiation. Experimental results showed that brain organoids induced with the Matrigel provided by the present invention exhibited an intact overall structure, with clear boundaries and a dense structure, indicating good intercellular adhesion. Organoids proliferated and migrated in the Matrigel, forming a wreath-like protrusion structure with a large diameter, reflecting good migration ability. Electrophysiological testing of the induced brain organoids revealed relatively active neurons, indirectly reflecting the high developmental maturity of the organoids and the high biological activity of the Matrigel provided by the present invention. In addition, the components of the matrix gel provided by the present invention are clear and distinct, with fewer uncertain components of animal origin. The standardized preparation can reduce batch differences, improve experimental repeatability and data reliability, and is suitable for large-scale research and has broad application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is a photograph of the matrix gel of Formula 1 in Example 1 after preparation (4°C, liquid state).

[0017] Figure 2 This is the state of the matrix gel of Formula 1 in Example 1 after solidification under the bright field vision (37° C., solid state).

[0018] Figure 3 These are electron micrographs of brain organoids induced to differentiate by matrix gel of formula 1 and formula 2 at different differentiation stages. DETAILED DESCRIPTION

[0019] To address the problems of existing hydrogels or Matrigel used to differentiate stem cells into brain organoids, such as large batch-to-batch variability, unclear composition, and / or poor bioactivity, the present invention aims to provide a hydrogel with a clear composition and excellent bioactivity, while reducing batch-to-batch variability in pure animal-derived materials. Furthermore, the results of the examples demonstrate that the Matrigel provided by the present invention can improve the differentiation efficiency of brain organoids, enhance the stability of the experimental system, and reduce costs.

[0020] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0021] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are to be considered as illustrative in nature and not restrictive.

[0022] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of the biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0023] Example 1: Matrigel for brain organoid differentiation and its preparation

[0024] The matrix gels of formulas 1-8 listed in Table 1 below for brain organoid differentiation were prepared respectively according to the following methods, wherein in Table 1, Neurobasal+B27 is a brain-like culture medium (wherein Neurobasal is a commercial culture medium, brand: Gibco, product number: 21103049; B27 is a commercial culture medium additive, brand: Gibco, product number: 17504044), which represents Neurobasal culture medium supplemented with 2% (volume percentage) B27, providing nutrients and growth factors required for cell growth and differentiation; GFR represents GFR matrix gel (Corning, 356231), and Matrigel represents Matrigel matrix gel (Corning, 354277), both of which can be used to provide the basic scaffold structure and extracellular matrix components required for cell growth; type I collagen represents PureCol ® Type I Collagen Solution, a high-purity bovine collagen solution produced by Advanced BioMatrix (Cat. No. 5005), is used to enhance the mechanical strength and cell adhesion of the matrix gel. β-Mercaptoethanol (Gibco, Cat. No. 21985-023) acts as an antioxidant to protect cells from oxidative stress and preserve the bioactive components of the experimental system.

[0025] In the formulation shown in Table 1 below, Cultrex ® BME (Cultrex(R) BME, Type 2 PathClear(R), Sigma, 3532-001-02) was used as the Matrigel component; IV collagen (Sigma, CAS No. 9007-34-5, 0.3 mg / ml); glutathione (GSH) (Sigma, 70-18-8); ascorbic acid (Sigma, A2218); and vitamin E (Sigma, 10191-41-0). In the formulation, 1 mM glutathione (GSH), 100 μM ascorbic acid, or 10 μg / mL vitamin E were used as antioxidants to compare the antioxidant effect of 1% β-mercaptoethanol.

[0026] The preparation method of formula 1, 3-8 is as follows: according to the proportion of matrix (GFR, Matrigel or Cultrex ® Mix BME and collagen (type I collagen or type IV collagen) under sterile conditions; then add antioxidants (β-mercaptoethanol, glutathione (GSH), ascorbic acid or vitamin E) and continue stirring until completely dissolved; finally add brain-like culture medium (Neurobasal + B27), mix thoroughly, and store at 4°C until ready for use. Figure 1 As shown in FIG, which exemplifies the state of Formula 1 after preparation (4°C, liquid state), the state of the matrix gel of Formula 1 after solidification observed under the microscope bright field field (37°C, solid state) is shown in FIG. Figure 2 shown.

[0027] Table 1: Matrigel formulation for brain organoid differentiation (% represents volume percentage)

[0028]

[0029] Example 2: Using Matrigel to Induce Differentiation of Brain Organoids

[0030] This example uses the Matrigel of Formulas 1-8 prepared in Example 1 to perform brain organoid differentiation culture. The specific operation method includes the following steps:

[0031] 2.1. Reagents and raw materials

[0032] hiPSCs: hiPSC-WL4802 (provided by Guodian (Beijing) Pharmaceutical Technology Co., Ltd.).

[0033] The brain organoid differentiation kit was provided by Guodian (Beijing) Pharmaceutical Technology Co., Ltd. (Cat. No.: GDK012).

[0034] Unless otherwise specified, other reagents and raw materials can be obtained from commercial sources. Experimental methods in the present invention without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0035] 2.2. Induced differentiation of brain organoids

[0036] 2.2.1. Culture of hiPSCs

[0037] hiPSCs were plated at 5-10 × 10 4Cells were seeded at 1:1 ratio per well in a six-well plate pretreated with Matrigel (Stem Cell) and cultured with mTeSR Plus (Stem Cell). After the confluence reached 70%-80%, they were washed once with preheated DPBS (Dulbecco's phosphate-buffered saline), digested with 0.5 mM EDTA (Invitrogen), passaged at a ratio of 1:6, and incubated in a 37°C, 5% CO2 incubator.

[0038] 2.2.2. Differentiation of hiPSCs into brain organoids

[0039] (1) Formation of embryoid bodies (Day 0-Day 5)

[0040] (1.1) Day 0: Remove the hiPSC medium (mTeSR Plus) and wash once with 1 mL of DPBS. Add 1 mL of Gentle Cell Dissociation Reagent (Stem Cell) to each well of the 6-well plate and return the cells to the incubator for 8-10 minutes. Add 1 mL of hiPSC medium and transfer the cell suspension to a 15 mL centrifuge tube. Rinse the wells with 1 mL of medium and transfer to the same 15 mL centrifuge tube. Centrifuge at 300 g for 5 minutes, discard the supernatant, and resuspend the cells in 1 mL of Seeding Medium (derived from the Brain Organoid Differentiation Kit GDK012). Count the cells using trypan blue and a hemocytometer. Add 150 μL of the cell suspension to each well of a U-bottom 96-well ultra-low attachment cell culture plate (Corning). Centrifuge the plate at 100 g for 3 minutes and incubate in a 37°C, 5% CO2 incubator for 24 hours to allow embryoid bodies to form.

[0041] According to the manual, the seeding medium consists of basal medium 1, supplement 1, and 10 μM Y-27632 in the brain organoid differentiation kit GDK012.

[0042] (1.2) Day 1: No need to change the cell culture medium. Gently place the cell culture plate under a microscope to observe the morphology and diameter of the embryoid bodies (EBs). The appropriate EB diameter range at this point is 100-200 μm.

[0043] (1.3) On Day 2, gently aspirate approximately 140 μL of culture medium and add 150 μL of fresh Medium 1 (complete Medium 1 in the Brain Organoid Differentiation Kit GDK012) without Y-27632. If there are many dead cells around the EBs, continue to add Y-27632 when changing the medium on Day 2. Change the medium every other day and culture until Day 5. Observe the EBs under a microscope on Day 5. The diameter should be greater than 300 μm, preferably 400-600 μm, and the edges should begin to become shiny and smooth before proceeding to the next step.

[0044] (2) Induction of brain organoids (Day 6-Day 10)

[0045] On Day 6, remove Medium 1 and add 200 μL of Medium 2 (complete medium 2 in the brain organoid differentiation kit GDK012) to each well. Place the cells in a 37°C, 5% CO2 incubator and culture until Day 10, changing the medium every other day.

[0046] According to the manual, Medium 2 consists of Basic Medium 2, Supplement 2-1, and Supplement 2-2 in the Brain Organoid Differentiation Kit GDK012.

[0047] (3) Formation of brain organoids (Day 11-Day 15)

[0048] (3.1) Day 11: Thaw the Matrigel of each formulation in Example 1 on ice for 1-2 h until completely melted. Place the Organoid Embedding Sheet (Stem Cell) in a clean 10 cm dish.

[0049] According to the manual, the Organoid Embedding Sheet can be replaced with parafilm sealing film.

[0050] (3.2) Use a wide-mouth 200 μL pipette tip to transfer EBs to the Organoid Embedding Sheet one by one.

[0051] According to the manual, do not transfer more than 16 EBs at a time to avoid EB shrinkage and premature failure of Matrigel polymerization.

[0052] (3.3) Add 15 μL of the formulated Matrigel to each EB, ensuring that the Matrigel fully covers the EB. Use a 10 μL pipette tip to adjust the position of the EB so that it is located in the center of the Matrigel.

[0053] (3.4) Place the 10 cm dish in an incubator for 30-45 minutes to allow the Matrigel to solidify.

[0054] (3.5) Add 3 mL of Medium 3 (complete Medium 3 in the Brain Organoid Differentiation Kit GDK012) to each well of a 6-well ultra-low attachment plate. Use forceps in your left hand to lift a corner of the sealing film. Use a pipette in your right hand to aspirate the medium and gently rinse to allow the Matrigel droplet and EBs to detach from the Organoid Embedding Sheet and transfer to the well containing medium below. Incubate in a 37°C, 5% CO2 incubator for 3 days.

[0055] According to the manual, Medium 3 consists of basal culture medium 3, additive 3-1, additive 3-2, and additive 3-3. Each well of a 6-well plate can culture 8-10 organoids. Once outward-expanding neuroepithelial buds are formed, the next step can be carried out.

[0056] (4) Maturation of brain organoids (Day 16-Day 40)

[0057] On Day 16, slowly remove the culture medium and gently add 3 mL of Medium 4 (complete medium 4 in the brain organoid differentiation kit GDK012) to each well. Place the cell culture plate on a horizontal shaker at 65-85 rpm and continue culturing in a 37°C, 5% CO2 incubator until Day 40. Measure the diameter of the brain organoids and collect them for MEA detection.

[0058] According to the manual, Medium 4 consists of basal medium 3, supplements 3-4, supplements 3-5, and supplements 3-6.

[0059] Electron microscopic observation of the brain organoids obtained by inducing differentiation using the various formulations in Example 1 showed that the brain organoids induced and differentiated using the Matrigel of Formulations 1, 2, and 3 grew relatively fully (however, there were large batch differences in the brain organoids induced and differentiated using the Matrigel of Formulations 2 and 3, as detailed below), while the brain organoids induced and differentiated using the Matrigel of Formulations 4 to 8 all showed insufficient growth and smaller brain organoid diameters (detailed below). Figure 3 As shown, it exemplarily shows electron microscopic photos of the matrix gels of formula 1 and formula 2 at different differentiation times during the differentiation and culture of brain organoids.

[0060] Some key indicators of formulas 1-8, including average material cost, mean brain-like diameter, batch standard deviation of brain-like diameter, and number of electrophysiological spikes (which can directly reflect the excitability of neurons in brain organoids and the level of network activity; the larger the number, the more active the neurons, and can also indirectly reflect the developmental maturity of brain organoids), were compared.

[0061] When evaluating the average material cost of each formulation of Matrigel, the total material cost was used for calculation.

[0062] When calculating the mean diameter of brain-like cells, 10 cells from each group of the same batch of induced differentiation were taken for diameter measurement and the mean value was calculated.

[0063] When calculating the batch standard deviation of brain-like diameter, each group used 5 batches of matrix gel for brain-like differentiation, and 10 brain-like cells were taken from each batch to calculate the average diameter, and then the standard deviation of the mean of the 5 batches was calculated.

[0064] To calculate the mean electrophysiological spike count, six cells from each group in the same batch of brain tissue were subjected to electrophysiological testing (MEA testing). The mean number of spikes detected within one minute was calculated, including the following steps:

[0065] Take Day 40 brain tissue for MEA detection:

[0066] (1) Matrigel coating: Dilute Matrigel with DMEM / F12 (Gibco, catalog number: 11320082) at a ratio of 1:50 and add 10 μL / well to the central area of ​​a 24-well MEA electrophysiological assay plate (Axion BioSystems). Sterile water or DPBS is added to the grooves between the wells to prevent evaporation of the coating solution.

[0067] (2) Fixation: Use a wide-mouth 200 μL pipette tip to transfer the brain organoids to the corresponding wells. Transfer the MEA plate to a 37°C incubator and place it for 2 h. Then add 500 μL of Medium 4 to each well. After overnight incubation, electrical activity can be recorded.

[0068] (3) MEA detection: Turn on the Maestro MEA system, transfer the MEA plate into the system and equilibrate for 10 min. Set the parameter to spontaneous, start recording the electrical activity of brain organoids for 5 min, and export the corresponding spike data for statistical analysis.

[0069] The test results of some key indicators of formulas 1-8 (including average material cost, mean brain-like diameter, batch standard deviation of brain-like diameter, and number of electrophysiological spikes) are shown in Table 2 below.

[0070] Table 2: Comparison of key indicators of different matrix gel formulations

[0071]

[0072] As shown in Table 2, compared to Formulations 2-4, Formulations 1 and 5-8 have lower average material costs, reducing costs by at least 28%. The mean diameter of brain organoids induced with Formulations 1-3 is larger than that of Formulations 4-8. This indicates that Formulations 1-3 resulted in adequate growth, while Formulations 4-8 showed inadequate growth, particularly when Formulations 6-9 (which differ from Formulation 1 only in the antioxidant) were used. The batch standard deviation of diameter for brain organoids induced with Formulation 1 is significantly smaller than that of Formulations 2-8, indicating that Formulation 1 provides the most consistent results. The mean number of electrophysiological spikes in brain organoids induced with Formulations 1-2 is significantly higher than that of Formulations 3-8, indicating that the neuronal excitability and network activity in brain organoids induced with Formulations 1-2 are higher, indicating greater neuronal activity and indirectly reflecting greater developmental maturity of brain organoids. From the results in Table 2, it is clear that the matrix gel of Formula 1 is significantly better in inducing the differentiation of brain organoids than Formulas 2-8. The various components in the matrix gel of Formula 1 can cooperate with each other to achieve a synergistic effect.

[0073] Example 3: Preparation of Matrigel

[0074] In Example 3, the Matrigel preparation method described in Example 1 was followed, and Matrigel with formulas 9-12 was also prepared as shown in Table 3 below.

[0075] Table 3: Matrigel formulation (% represents volume percentage)

[0076]

[0077] According to the operating procedures for inducing differentiation and culturing brain organoids described in Example 2, the formulas in Table 3 above were used to induce differentiation and culture of brain organoids, and the key indicators of the brain organoids induced by each formula (including the mean brain organoid diameter, the batch standard deviation of the brain organoid diameter, and the mean number of electrophysiological spikes) were tested and calculated. The results are shown in Table 4 below.

[0078] Table 4: Comparison of key indicators of different matrix gel formulations

[0079]

[0080] The results in Table 4 above show that, similar to the results of Formulation 1, the mean diameter and mean number of electrophysiological spikes obtained by induced differentiation using Formulations 10-13 were also higher, and the batch standard deviation of brain organoid diameter was also lower, indicating that the Matrigel of Formulations 10-13 can also be used for brain organoid differentiation, and has better results than Formulations 2-8. Although the components of Formulations 9 and 14 are the same as those of Formulations 1 and 10-13, because the content is not within the appropriate range, the diameter of the induced differentiated brain organoids is smaller (indicating insufficient growth), the batch standard deviation of brain organoid diameter is also larger, and the mean number of electrophysiological spikes is lower.

[0081] In summary, the results indicate that the optimized matrix gel formula for brain organoid differentiation of the present invention may comprise or consist of 20%-40% GFR, 50%-70% type I collagen, 0.5%-2% β-mercaptoethanol, and 8%-9.5% Neurobasal+B27 (meaning Neurobasal culture medium supplemented with 2% (volume percentage) B27), preferably 25%-35% GFR, 55%-65% type I collagen, 1%-1.5% β-mercaptoethanol, and 8.5%-9% Neurobasal+B27, and more preferably 30% GFR, 60% type I collagen, 1% β-mercaptoethanol, and 9% Neurobasal+B27.

[0082] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A matrix gel, which is composed of the following components in percentage by volume: 20%-40% GFR Matrigel 356231, 50%-70% type I collagen, 0.5%-2% beta-mercaptoethanol, and 8%-9.5% Neurobasal+B27; The Neurobasal+B27 refers to a Neurobasal culture medium supplemented with 2% by volume of B27.

2. The matrix gel according to claim 1, which is composed of the following components in percentage by volume: 25%-35% GFRMatrigel 356231, 55%-65% type I collagen, 1%-1.5% beta-mercaptoethanol, and 8.5%-9% Neurobasal+B27, or consisting thereof.

3. The matrix gel according to claim 1, which is composed of the following components in percentage by volume: 30% GFRMatrigel 356231, 60% type I collagen, 1% beta-mercaptoethanol, and 9% Neurobasal+B27, or consisting thereof.

4. The method for preparing the matrigel according to any one of claims 1 to 3, comprising the following steps: S1: Mix GFR Matrigel 356231 and type I collagen in the correct proportions under sterile conditions. S2: adding β-mercaptoethanol to the product obtained in step S1 and stirring until completely dissolved; and S3: Add Neurobasal+B27 to the product obtained in step S2 and mix thoroughly to obtain the matrix gel.

5. Use of the matrigel according to any one of claims 1 to 3 in inducing stem cells to differentiate into brain organoids; The stem cells are induced human pluripotent stem cells.

6. A method for inducing stem cells to differentiate into brain organoids, comprising embedding embryoid bodies formed by stem cells using the matrigel according to any one of claims 1 to 3, and culturing them, wherein the stem cells are induced human pluripotent stem cells.

7. A brain organoid obtained by the method according to claim 6.

8. Use of the brain organoids according to claim 7 in developmental biology research, disease models and drug screening, wherein the use is for non-disease diagnosis and treatment purposes.

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