WO2023160688A1 - 一种大脑类器官模型及其制备方法与应用 - Google Patents

一种大脑类器官模型及其制备方法与应用 Download PDF

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WO2023160688A1
WO2023160688A1 PCT/CN2023/078353 CN2023078353W WO2023160688A1 WO 2023160688 A1 WO2023160688 A1 WO 2023160688A1 CN 2023078353 W CN2023078353 W CN 2023078353W WO 2023160688 A1 WO2023160688 A1 WO 2023160688A1
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stem cells
pluripotent stem
brain
brain organoid
organoid model
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French (fr)
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陈璞
李彬
尚佳
赵稳
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武汉大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • G—PHYSICS
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    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the technical fields of stem cells, organoids, organoid chips and tissue engineering, in particular to a brain organoid model and its preparation method and application.
  • the brain is the most complex organ in human body structure and function. Understanding human brain development and diseases is one of the biggest challenges in life sciences. However, the difficulty of obtaining human brain tissue has seriously hindered our pace of deciphering the secrets of the human brain.
  • researchers have long used cell culture and animal models to study adult brain development and disease. These studies provide the foundation for our current understanding of brain development and function. Nonetheless, our understanding of the human brain is limited to simple cell-cell interactions and features shared by humans and vertebrates. Therefore, in order to further study the mechanism of human brain development and explore the pathogenesis and treatment of brain-related diseases, it is of great significance to establish an in vitro brain research model that is highly related to the human body.
  • Cerebral organoids are an emerging brain research model that has key features of the human brain, such as multiple brain-specific cell types, apical-basal polarity, division of neural stem cells, and neuronal migration patterns.
  • the brain organoid model has no species difference, and has structures and cell types that are highly related to the human body; compared with the two-dimensional culture model, it has a similar cellular microenvironment to the body and a variety of brain cells Population, the ability to simulate neural electrical signals in the brain and other advantages. Therefore, the construction of brain organoids in vitro provides an effective model system for studying human brain development and diseases.
  • the mainstream brain organoid culture protocol is this culture protocol or its variants, mainly including four stages: embryoid body formation, neuroectoderm induction, neuroepithelial differentiation and Brain organoids mature. Since the monolayer of adherent cells will be evenly exposed to differentiation factors during the growth process, it is difficult to produce a heterogeneous self-organized structure due to the concentration gradient of the factors and the interaction between cells, so in the process of brain organoid culture , it is necessary to form embryoid body pellets from stem cells in a low-adhesion U-bottom well plate or by the hanging drop method, and then coat the embryoid body after neural induction with Matrigel, and then transfer to a low-adhesion culture Dynamic culture in plates or bioreactors.
  • organoids are affected by external stimuli from human manipulation and location uncertainty, and are easily contaminated or fused, and difficult to locate and observe.
  • the above-mentioned limitations lead to a complex, highly variable, low-throughput and difficult-to-monitor organoid culture process.
  • the purpose of the present invention is to provide a brain organoid model and its preparation method and application, which can realize high-throughput, one-step construction of brain organoids with uniform shape, and can observe the whole process of organoid growth and development in situ.
  • a method for preparing a brain organoid model comprising:
  • Adding EB formation medium to the pluripotent stem cells with border-limited monolayer adherent growth to maintain culture for 4-6 days, using neural induction medium for 2-6 days, and then aspiration and discarding the neural induction medium for culture Add pre-cooled Matrigel for coating, and after solidification, replace the neural differentiation medium and culture for 3 to 5 days, and culture for 10 to 30 days in the induction maturation medium to obtain the brain organoid model.
  • the method for obtaining border restriction in monolayer adherent growth of pluripotent stem cells with border restriction includes one of molecular stamp method, photolithography method, perforated film method and differential adhesion method.
  • the obtaining pluripotent stem cells with border-limited monolayer adherent growth specifically includes:
  • Pluripotent stem cells growing adherently in a monolayer with boundary restrictions.
  • the outer dimension of the film matches the cell culture plate;
  • the shape of each perforated hole of the film includes circle, ellipse, semicircle, sector, triangle, quadrangle, pentagon, One of hexagonal and arbitrary polygonal;
  • the arrangement of perforations in the film includes: one of square apex arrangement, regular hexagonal arrangement, linear arrangement and random arrangement.
  • the cell adhesion material includes at least one of Matrigel and Vitronectin; the concentration of the cell adhesion material is 0.1-1 mg/mL.
  • the pluripotent stem cells include one of human embryonic stem cells or human induced pluripotent stem cells;
  • the growth area of the boundary-restricted single-layer adherent growth of pluripotent stem cells is 0.005-3.5mm 2
  • the distance between two adjacent boundary-limited single-layer adherent growth of pluripotent stem cells is 0.5-245mm.
  • the density of the boundary-restricted pluripotent stem cells when added to the EB formation medium is 1 ⁇ 10 5 -1 ⁇ 10 6 (cells/cm 2 ).
  • the shape of the generated brain organoid is spherical, that is, the shape of a ball cut off by a plane, and the neural rosette-like structure is randomly distributed around the periphery;
  • the brain organoid includes but is not limited to neurons, neural stem cells, star Brain-associated cell type called glia, microglia.
  • the culture method of the induction maturation medium is dynamic culture on a rocker shaker with an inclination angle ⁇ 25°.
  • the cell culture plate includes one of a 384-well plate, a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, and a 6-well plate.
  • the thickness of the bottom of the cell culture plate is 0.1-1.3 mm.
  • the obtaining of pluripotent stem cells with border-limited monolayer adherent growth specifically includes:
  • Pluripotent stem cells growing adherently in a monolayer with boundary restrictions.
  • the cell culture dish includes one of a 3.5 cm culture dish, a 6 cm culture dish and a 10 cm culture dish.
  • a brain organoid model obtained by the method is provided.
  • the application of the brain organoid model in neural mechanism research, neurological disease model, neuropharmaceutical development and neurotoxicity analysis is provided.
  • a brain organoid model provided by the present invention and its preparation method and application limit the growth of pluripotent stem cells on a two-dimensional plane through boundaries, and induce differentiation toward nerves, and finally form a brain organoid model with a thickness of 500-1500 ⁇ m and
  • the brain organoid model with a specific shape can realize high-throughput and one-step construction of organoids with uniform shape, and can observe the whole process of organoid growth and development in situ;
  • the brain organoids in the present invention can be analyzed in situ in a well plate without destroying the organoid structure, and are suitable for existing biological analysis and imaging instruments (such as high-content instruments) used in the field of drug development. Have good compatibility.
  • the brain organoid model of the present invention has obvious differences and advantages compared with the existing methods for culturing brain organoids, which are embodied in: the classic brain organoid culture methods all need to spontaneously aggregate suspended single cells into pseudo Embryoid body pellets, it is difficult to control the amount of cells in the final pellet at this stage; the formed embryoid bodies need to be transferred to different well plates multiple times; brain organoids are dynamically cultured in suspension, and it is difficult to observe the culture status of organoids in real time, etc. is an obstacle to the standardization of brain organoids.
  • the present invention directly utilizes the pluripotent stem cells cultivated in a single layer of adherent cells, and limits its growth range through boundaries, creating a brain organoid model and its preparation method for adherent growth.
  • the relevant parameters of the preparation method have been determined.
  • the formation position, shape, and size are highly uniform, and the throughput is high, which is easy for in-situ observation and real-time imaging.
  • the model and production method created by the present invention are significantly different, and have irreplaceable Compared with other advantages, it solves the problems of complex culture of brain organoids, low throughput, and difficulty in in situ observation.
  • Fig. 1 is a schematic diagram of making a culture chip of brain organoids provided by an embodiment of the present invention
  • Figure 2a is a patterned chip
  • Figure 2b is a bright-field picture of patterned brain organoids
  • Figure 2c is the result of comparing the area of patterned and traditionally cultured brain organoids at day 25, and Figure 2d is at day 25 The results of comparing the coefficient of variation of the area of brain organoids cultured by patterning and traditional methods;
  • Figure 3 is the real-time monitoring results of the development of patterned brain organoids under bright field conditions
  • Figure 4 shows the development of patterned brain organoids detected at the protein level by immunofluorescence and frozen section methods, and the expression of MAP2 gene after treatment with different concentrations of A ⁇ 42 O, the scale bar is 100 ⁇ m.
  • Figure 5 shows the monitoring results of the development of patterned brain organoids of different sizes under bright field conditions, and the scale bar is 100 ⁇ m.
  • Figure 6 shows the death of neurons after treatment with 5 ⁇ M A ⁇ 42 O.
  • the staining method is TUNEL staining, and the scale bar is 50 ⁇ m.
  • a method for preparing a brain organoid model comprising:
  • Step S1 obtaining pluripotent stem cells growing adherently in a monolayer with boundary restrictions
  • the method for obtaining the border restriction in the monolayer adherent growing pluripotent stem cells with border restriction includes one of molecular stamp method, photolithography method, perforated film method and differential adhesion method.
  • the perforated film method is adopted, and the specific operations are as follows:
  • Step S101 obtaining a film with multiple perforations
  • step S101 specifically includes:
  • Step S1011 obtaining a positive mold with a micropillar array; the height of the micropillar array of the positive film is 30 ⁇ m ⁇ 100 ⁇ m. If the height is too low, the film will be too thin and difficult to handle; if the height is too high, it will not be easy to perforate.
  • Step S1012 pour PDMS onto the positive mold, vacuum dry and vacuumize, and cover a layer of PMMA on the PDMS, then clamp and fix it with two glass sheets, and dry;
  • Step S1013 taking out the solidified PDMS layer to obtain a film with multiple perforations.
  • the material of the film may specifically be polydimethylsiloxane.
  • the external dimensions of the film match the cell culture plate
  • the hole shape of each perforation of the film includes one of circle, ellipse, semicircle, sector, triangle, quadrangle, pentagon, hexagon and any polygon;
  • the arrangement of perforations in the film includes: one of square apex arrangement, regular hexagonal arrangement, linear arrangement and random arrangement.
  • each perforation of the film is 0.005-3.5 mm 2 , and the distance between two adjacent perforations of the film is 0.5-245 mm. If the perforation area is less than 0.005mm 2 , there is a disadvantage that it is easy to lose when changing the liquid; if the area is too large, it is difficult to form a brain organoid; if the distance between two adjacent perforations of the film is less than 0.5mm, there is a disadvantage of organoid fusion , if the spacing is too large, it cannot be adapted to the existing cell culture system; multiple perforations can be arranged regularly or irregularly.
  • the cell culture plate includes one of a 384-well plate, a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, and a 6-well plate.
  • the cell culture dish When a cell culture dish is used, the cell culture dish includes one of a 3.5 cm culture dish, a 6 cm culture dish and a 10 cm culture dish.
  • Step S102 placing the film on the bottom of the cell culture plate, and adding a cell adhesion material to the cell culture plate for coating;
  • the cell adhesion material includes at least one of Matrigel and Vitronectin; the concentration of the cell adhesion material is 0.1-1 mg/mL. If the concentration is too low, there is a disadvantage that cells are difficult to attach; if the concentration is too high, it is difficult to remove the perforated film, and the effect of cell patterning is not good.
  • Step S103 inoculating pluripotent stem cells into the coated cell culture plate, and removing the thin film after the cells adhere to the wall, so as to obtain pluripotent stem cells growing adherently in a single layer with boundaries.
  • the method for obtaining boundary restrictions in pluripotent stem cells growing adherently in a monolayer with boundary restrictions includes molecular stamp method, photolithography method, perforated film method and differential adhesion method. kind of. Other methods can also be used to obtain pluripotent stem cells with boundary-limited single-layer adherent growth. It is only necessary to control the growth area of the boundary-limited single-layer adherent growth of pluripotent stem cells to 0.005-3.5mm 2 , adjacent The distance between the two regions of pluripotent stem cells growing adherently in a single layer with boundaries is 0.5-245mm.
  • the geometric shape limited by the boundary includes one of circle, ellipse, semicircle, sector, triangle, quadrilateral, pentagon, hexagon and any polygon; the arrangement of pluripotent stem cells limited by the boundary includes: One of square vertex arrangement, regular hexagonal arrangement, linear arrangement and arbitrary arrangement.
  • the seeding density of the human pluripotent stem cells is 1 ⁇ 10 5 -1 ⁇ 10 6 (cells/cm 2 ).
  • the pluripotent stem cells include one of human embryonic stem cells and human induced pluripotent stem cells, and the seeding density is 10 5 -10 6 cells per square centimeter. If the seeding density is too low, it will be difficult for the cells to fuse in a short time, and it is difficult to become a brain organoid with a certain thickness; if the density is too high, it will affect the state of the cells when they start to differentiate, and there will be too many dead cells during the culture process.
  • Step S2 adding EB formation medium to the pluripotent stem cells with border-limited monolayer adherent growth to maintain culture for 4-6 days, culturing with neural induction medium for 2-6 days, and then discarding the neural induction medium.
  • pre-cooled Matrigel was added for coating, and after solidification, the neural differentiation medium was replaced sequentially for 3 to 5 days, and the induction maturation medium was cultured for 10 to 30 days to obtain the brain organoid model.
  • the neural induction medium was used to culture for 2 to 6 days, in order to induce the cells to differentiate into neuroectoderm and continue to proliferate;
  • the basic component of the EB formation medium is Nuwacell TM hiPSC/hESC medium-ncTarget, and additionally add ROCK inhibitor Y27632 and bFGF;
  • the mother solution concentration of the ROCK inhibitor Y27632 is 10 mM;
  • the volume ratio of the mother solution of ROCK inhibitor Y27632 to ncTarget is 1:1000;
  • the final concentration of bFGF is 2-6 ng/mL, preferably 4 ng/ml.
  • the basic component of the neural induction medium is DMEM/F12, and in addition, 1% NEAA (Non Essential Amino Acid, 100 ⁇ ) and 1% GlutaMAX (100 ⁇ ) (available at GIBCO/Invitrogen Cat. No. 35050061), total volume 1% N2 (100 ⁇ ) (N-2 additive), 0.5-2 ⁇ g/mL heparin (preferably 1 ⁇ g/ml), 1% penicillin-streptomycin (100 ⁇ ) in the total volume.
  • NEAA Non Essential Amino Acid, 100 ⁇
  • GlutaMAX 100 ⁇
  • N-2 additive total volume
  • heparin preferably 1 ⁇ g/ml
  • penicillin-streptomycin 100 ⁇
  • the basic components of the neural differentiation medium are DMEM/F12 and Neurobasal medium, the volume ratio of which is 1:1, and 1% B27 without Vitamin A (50 ⁇ ) accounting for the total volume needs to be added, accounting for 0.5% N2 (100 ⁇ ) in total volume, 0.5% NEAA (100 ⁇ ) in total volume, 1% GlutaMAX (100 ⁇ ) in total volume, 1% penicillin-streptomycin (100 ⁇ ) in total volume, 2 ⁇ 5 ⁇ g/ml beta-mercaptoethanol, preferably 3.5 ⁇ g/ml, 1-4 ⁇ g/ml insulin solution, preferably 2.5 ⁇ g/ml.
  • the basic components of the induction maturation medium are DMEM/F12 and Neurobasal medium, and its volume ratio is 1:1.
  • 1% B27 (50 ⁇ ) of the total volume needs to be added, accounting for the total volume.
  • the diameter reaches 3-4 mm.
  • the interior of brain organoids contains a large number of cells that are hypoxic and necrotic.
  • the patterned brain organoids constructed by this method can greatly reduce internal cell necrosis.
  • a cell-specific adhesion area with a specific pattern is formed on the bottom of the cell culture vessel through patterning technology.
  • Pluripotent stem cells can be inoculated on the patterned substrate. After the cells adhere to the wall, the perforated film is removed to obtain a specific edge shape. Cell aggregates on this patterned substrate induce stem cells to differentiate into brain organoids, and finally form a brain organoid model with a thickness of 500-1500 ⁇ m and a specific edge shape, with high uniformity and high throughput, suitable for cell-based culture A range of imaging and analysis instruments for well plates.
  • a brain organoid model obtained by the method is provided.
  • the obtained brain organoid is spherical, that is, the shape of a ball cut off by a plane, which is different from the phenotype of the brain organoid; the nerve flowers are randomly distributed in the periphery Ring-like structures, and occasionally vacuoles around some brain organoids.
  • an application of the brain organoid model in neurotoxicity analysis is provided.
  • the preparation method of the brain organoid model comprises the acquisition of a perforated film, the preparation of a patterned chip, and the preparation of pluripotent stem cells Seeded on a patterned substrate, differentiated towards the neuroectoderm, and finally formed a spherical segmental brain organoid that adhered to the bottom of the culture dish and had a certain thickness and a specific edge shape.
  • the cell types contained in it included stem cell-derived neural stem cells, Neurons, astrocytes, and microglia; the brain organoids can be applied to the study of brain development, disease, drug screening, drug neurotoxicity evaluation, and the like.
  • the present invention provides a high-throughput, high-uniform, in situ cultured brain organoid model, providing an innovative research tool for related brain organoid research.
  • Embodiment 1 A kind of culture method of brain organoid model
  • the SU-8 template was prepared using soft lithography technology.
  • This template is a high-throughput microcolumn array with a microcolumn diameter of 500 microns, a height of 50 microns, and a microcolumn spacing of 1000 microns;
  • the boundary restriction shape of the pluripotent stem cells is circular
  • the growth area of the pluripotent stem cells (that is, the area of each perforation of the film) is 0.2 mm 2 ;
  • the distance between the growth regions of the pluripotent stem cells (that is, the distance between two adjacent perforations of the film) is 1.5mm;
  • the growth regions of the pluripotent stem cells are arranged in a square apex arrangement
  • the culture system of the brain organoid is a 48-well plate.
  • the boundary shape of the pluripotent stem cells is circular; the growth area of the pluripotent stem cells is 0.008 mm 2 ; the distance between the growth regions of the pluripotent stem cells is 1 mm; the growth regions of the pluripotent stem cells are arranged
  • the method is that the vertices of the square are arranged; the culture system of the brain organoid is a 48-well plate; the others are the same as in Example 1.
  • the boundary shape of the pluripotent stem cells is circular; the growth area of the pluripotent stem cells is 0.03mm 2 ; the distance between the growth regions of the pluripotent stem cells is 1.2mm; the growth region of the pluripotent stem cells
  • the arrangement method is a square apex arrangement; the culture system of the brain organoids is a 48-well plate; others are the same as in Example 1.
  • the boundary shape of the pluripotent stem cells is circular; the growth area of the pluripotent stem cells is 0.8 mm 2 ; the distance between the growth regions of the pluripotent stem cells is 2 mm; the growth regions of the pluripotent stem cells are arranged
  • the method is that the vertices of the square are arranged; the culture system of the brain organoid is a 48-well plate; the others are the same as in Example 1.
  • the boundary shape of the pluripotent stem cells is a square; the growth area of the pluripotent stem cells is 1 mm 2 ; the distance between the growth regions of the pluripotent stem cells is 2 mm; the arrangement of the growth regions of the pluripotent stem cells is: The apexes of the square are arranged; the culture system of the brain organoid is a 48-well plate; the others are the same as in Example 1.
  • the boundary restriction shape of the pluripotent stem cells is an equilateral triangle; the growth area of the pluripotent stem cells is 0.43 mm 2 ; the growth area spacing of the pluripotent stem cells is 2 mm; the growth area of the pluripotent stem cells is arranged
  • the method is that the vertices of the square are arranged; the culture system of the brain organoid is a 48-well plate; the others are the same as in Example 1.
  • the boundary restriction shape of the pluripotent stem cells is a cross; the growth area of the pluripotent stem cells is 0.2 mm 2 ; the distance between the growth regions of the pluripotent stem cells is 1mm; the arrangement of the growth regions of the pluripotent stem cells is a square apex arrangement; the culture system of the brain organoids is a 48-well plate; others are the same as in Example 1.
  • the boundary shape of the pluripotent stem cells is circular; the growth area of the pluripotent stem cells is 0.2 mm 2 ; the distance between the growth regions of the pluripotent stem cells is 1 mm; the culture system of the brain organoids is 24 orifice plates; others are the same as in Example 1.
  • the boundary shape of the pluripotent stem cells is circular; the growth area of the pluripotent stem cells is 0.2 mm 2 ; the distance between the growth areas of the pluripotent stem cells is 1.5 mm; the perforation arrangement is regular hexagonal shape arrangement; the culture system of the brain organoids is a 24-well plate; others are the same as in Example 1.
  • the cell adhesion substance of the patterned brain organoid chip is Vitronectin, and the others are the same as in Example 1.
  • the restricted boundary area of the pluripotent stem cells is 0.002 mm 2 , and the others are the same as in Example 1.
  • the limited boundary area of the pluripotent stem cells was 5 mm 2 , and the others were the same as in Example 1.
  • the distance between the growth regions of pluripotent stem cells was 0.1 mm, and the others were the same as in Example 1.
  • the distance between the growth regions of the pluripotent stem cells was 0.05 mm, and the others were the same as in Example 1.
  • the cell density at which the brain organoids start to differentiate is 5 ⁇ 10 3 /cm 2 , and the others are the same as in Example 1.
  • the cell density at which the brain organoid begins to differentiate is 5 ⁇ 10 7 /cm 2 , and the others are the same as in Example 1.
  • the cell adhesion substance of pluripotent stem cells was 10 mg/mL Matrigel, and the others were the same as in Example 1.
  • the cell adhesion substance of the pluripotent stem cells in this comparative example was 5 mg/mL Matrigel, and the others were the same as in Example 1.
  • the cell adhesion substance of pluripotent stem cells was 0.01 mg/mL Matrigel, and the others were the same as in Example 1.
  • the limited boundary area of pluripotent stem cells is 0.002 mm 2 , which is smaller than the range of 0.005-3.5 mm 2 in the embodiment of the present invention, and there are disadvantages that the organoids are not tightly attached and are easily lost during the liquid exchange process;
  • the limited boundary area of pluripotent stem cells is 5 mm 2 , which is larger than the range of 0.005 to 3.5 mm 2 in the embodiment of the present invention, and there is a disadvantage that patterned brain organoids are difficult to form;
  • the distance between the growth regions of pluripotent stem cells is 0.1mm, which is smaller than the range of 0.5-245mm in the embodiment of the present invention, which will cause fusion during the growth and enlargement of organoids, and has the disadvantage of being difficult to form independent organoids;
  • the distance between the growth regions of pluripotent stem cells is 0.05 mm, which is 0.5-245 mm smaller than that of the embodiment of the present invention
  • the range of organoids will cause fusion during the growth and enlargement of organoids, and there is a disadvantage that it is difficult to form independent organoids;
  • the cell density at which brain organoids begin to differentiate is 5 ⁇ 10 3 /cm 2 , which is lower than the range of 10 5 to 10 6 in Examples of the present invention, and the initial cell density is too low to form organoids of various cell types. organ defects;
  • the cell density at which the brain organoid begins to differentiate is 5 ⁇ 10 7 /cm, which is greater than the range of 10 5 to 10 6 in the embodiment of the present invention, and has the disadvantage that the initial cell density is too high and affects the subsequent differentiation state;
  • the cell adhesion substance of pluripotent stem cells is 10 mg/mL Matrigel, which is greater than the range of 0.1-1 mg/mL in the embodiment of the present invention, and has the disadvantage of inducing early differentiation of cells in other directions;
  • the cell adhesion substance of pluripotent stem cells is 5 mg/mL Matrigel, which is greater than the range of 0.1-1 mg/mL in the embodiment of the present invention, and has the disadvantage of inducing early differentiation of cells in other directions;
  • the cell adhesion substance of pluripotent stem cells is 0.01 mg/mL Matrigel, which is less than the range of 0.1-1 mg/mL in the embodiment of the present invention, and there is a disadvantage that the concentration of extracellular adhesion substance is too low and the cells are difficult to attach;
  • Example 1 to Example 10 of the present invention brain organoid models with a thickness of 500-1500 ⁇ m and a specific edge shape were formed, with high uniformity.
  • the present invention utilizes boundary restriction on monolayer cultured pluripotent stem cells to generate a new type of brain organoid model in the form of adhering to the wall.
  • the model has neurons, neural stem cells, astrocytes, etc.
  • Brain-related cell types can achieve high-throughput and one-step construction of brain organoids with uniform morphology, avoiding the risk of fusion and contamination caused by methods and operations during organoid culture, and can observe the growth and development of organoids in situ The whole process, for the applied research of brain organoids.
  • Immunofluorescence detection of markers of different cell types in brain organoids Patterned brain organoids were fixed and sectioned at day 25 in culture. Co-staining of NEXTIN and SOX2 identified neural stem cell differentiation, TUJ and MAP2 identified neuronal differentiation, and PAX6 identified expression of forebrain-related markers in patterned brain organoids.
  • Example 2 Construction and characterization of the A ⁇ 42 oligomer neurotoxicity evaluation system on the patterned substrate of Example 1: On the 25th day, the A ⁇ 42 oligomers (A ⁇ 42 O) of Control, 0 ⁇ M, 1 ⁇ M, and 5 ⁇ M were used on the brain class respectively. 72 hours after organoid treatment, QPCR was performed to detect the expression of neuron-related markers. The result is shown in Figure 4, showing that the embodiment of the present invention is successful Human brain organoids were obtained and an evaluation system for neurotoxicity of A ⁇ 42 oligomers was constructed.
  • Example 4 Construction and characterization of the A ⁇ 42 oligomer neurotoxicity evaluation system on the patterned substrate of Example 1: On the 25th day, the brain organoids were treated with Control and 5 ⁇ M A ⁇ 42 oligomers ( A ⁇ 42O ) respectively. After 72 hours, TUNEL staining was performed to detect neuronal apoptosis. The results are shown in Figure 6, which shows that the embodiment of the present invention successfully obtained human brain organoids and constructed an A ⁇ 42 oligomer neurotoxicity evaluation system.

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Abstract

本发明公开了一种大脑类器官模型及其制备方法与应用，所述方法包括：获得具有边界限制的单层贴壁生长的多能干细胞，加入EB形成培养基维持培养4～6天；后采用神经诱导培养基培养2～6天；后吸弃培养基，用预冷的Matrigel包被，固化后，依次更换神经分化培养基培养3～5天、诱导成熟培养基培养10～30天，获得大脑类器官模型。

Description

一种大脑类器官模型及其制备方法与应用 技术领域
本发明涉及干细胞、类器官、类器官芯片与组织工程技术领域，特别涉及一种大脑类器官模型及其制备方法与应用。
背景技术
大脑是人全身结构和功能最复杂的器官，了解人类大脑发育和疾病是生命科学中最大的挑战之一。然而，人脑组织的获取困难严重阻碍了我们破解人类大脑秘密的步伐。一直以来，研究人员利用细胞培养模型和动物模型来研究成人类大脑发育与疾病。这些研究为我们目前对大脑发育和功能的理解奠定了基础。尽管如此，我们对人类大脑的理解仅限于简单细胞间的相互作用及人与脊椎动物共有的特征。因此，为了进一步研究人类大脑发育的机制，探索大脑相关疾病的发病原因与治疗方法，建立与人体高度相关的体外大脑研究模型具有重要意义。大脑类器官是一种新兴的大脑研究模型，具有人类大脑的关键特征，如多种大脑特异性细胞类型、顶-基底极性、神经干细胞的分裂和神经元迁移模式等。与传统动物模型相比，大脑类器官模型不存在种属差异，具有和人体高度相关的结构与细胞类型；与二维培养模型相比，具有与体内相似的细胞微环境、具有多种大脑细胞种群、能够模拟大脑中的神经电信号等优势。因此体外大脑类器官的构建为研究人类大脑发育和疾病提供了一个有效的模型系统。
自2013年Madeline Lancaster首次培养出大脑类器官后，主流的大脑类器官培养方案均为该培养方案或其变体，主要包括四个阶段：拟胚体形成、神经外胚层诱导、神经上皮分化和大脑类器官成熟。由于单层贴壁细胞在生长的过程中会均匀接触到到分化因子，从而难以由于因子的浓度梯度、细胞间相互作用等产生具有异质性自组织的结构，因此在大脑类器官培养过程中，需要先将干细胞在低粘附U型底孔板或采用悬滴法形成拟胚体小球，再将经过神经诱导后的拟胚体进行包被Matrigel，随后转入至低粘附的培养板或生物反应器中进行动态培养。然而，在类器官的转移及悬浮培养的过程中，类器官受到人为操作的外界刺激，以及位置不确定性的影响，极易污染或融合，并且难以定位观察。上述局限性导致了类器官的培养过程复杂、差异性大、通量低且不易于实时监测。现有的大脑类器官培养方法专利中，均需从细胞成球开始。
因此，为了更好的解决现有大脑类器官培养方法存在的问题，有必要开发一种原位高通量及高均一性大脑类器官模型，用于大脑类器官的应用研究。
发明内容
本发明目的是提供一种大脑类器官模型及其制备方法与应用，可实现高通量、一步法构建形态均一的大脑类器官，并可原位观察类器官生长、发育全过程。
为了实现上述目的，本发明采用如下技术方案：
在本发明的第一方面，提供了一种大脑类器官模型的制备方法，所述方法包括：
获得具有边界限制的单层贴壁生长的多能干细胞；
向所述具有边界限制的单层贴壁生长的多能干细胞中加入EB形成培养基维持培养4～6天，采用神经诱导培养基培养2～6天，后吸弃所述神经诱导培养基培养基，加入预冷的Matrigel进行包被，固化后，依次更换神经分化培养基培养3～5天、诱导成熟培养基培养10～30天，获得大脑类器官模型。
进一步地，所述获得具有边界限制的单层贴壁生长的多能干细胞中的边界限制的方法包括分子印章法、光刻法、穿孔薄膜法和差异性粘附法中的一种。
进一步地，所述获得具有边界限制的单层贴壁生长的多能干细胞，具体包括：
获得具有多个穿孔的薄膜；
将所述薄膜置于细胞培养板底部，并向所述细胞培养板中加入细胞粘附材料进行包被；
向所述包被后细胞培养板内接种多功能干细胞，待细胞贴壁后，将所述薄膜移走，获得
具有边界限制的单层贴壁生长的多能干细胞。
进一步地，所述薄膜外形尺寸与所述细胞培养板相匹配；所述薄膜的每个所述穿孔的孔形状包括圆形、椭圆形、半圆形、扇形、三角形、四边形、五边形、六边形和任意多边形中的一种；所述薄膜中穿孔的排列方式包括：正方形顶点排列，正六边形排列、线性排列和任意排列方式中的一种。
进一步地，所述细胞黏附材料包括Matrigel和Vitronectin中的至少一种；所述细胞黏附材料的浓度为0.1～1mg/mL。
进一步地，所述多能干细胞包括人胚胎干细胞或人诱导多能干细胞中的一种；
进一步地，所述具有边界限制的单层贴壁生长的多能干细胞的生长面积为0.005～3.5mm2，相邻两个所述具有边界限制的单层贴壁生长的多能干细胞区域的间距为0.5～245mm。
进一步地，所述具有边界限制的多能干细胞开始加入EB形成培养基时的密度为1×105～1×106(个/cm2)。
上述技术方案中，产生的大脑类器官形状为球缺形，即一个球被平面截下一部分后的形状，外周随机分布神经花环状结构；大脑类器官包括但不限于神经元、神经干细胞、星 形胶质细胞、小胶质细胞的大脑相关细胞类型。
上述技术方案中，所述诱导成熟培养基培养方式为倾斜角≤25°的翘板摇床动态培养。
上述技术方案中，所述细胞培养板包括384孔板、96孔板、48孔板、24孔板、12孔板、6孔板中的一种。
上述技术方案中，所述细胞培养板底部厚度为0.1-1.3mm。
上述技术方案中，所述获得具有边界限制的单层贴壁生长的多能干细胞，具体包括：
获得具有多个穿孔的薄膜；
将所述薄膜置于细胞培养皿底部，并向所述细胞培养皿中加入细胞粘附材料进行包被；
向所述包被后细胞培养皿内接种多功能干细胞，待细胞贴壁后，将所述薄膜移走，获得
具有边界限制的单层贴壁生长的多能干细胞。
上述技术方案中，所述细胞培养皿包括3.5cm培养皿、6cm培养皿和10cm培养皿中的一种。
在本发明的第二方面，提供了一种采用所述的方法获得的大脑类器官模型。
在本发明的第三方面，提供了所述的大脑类器官模型在神经机制研究、神经疾病模型、神经药物开发及神经毒性分析中的应用。
本发明实施例中的一个或多个技术方案，至少具有如下技术效果或优点：
(1)本发明提供的一种大脑类器官模型及其制备方法与应用，通过边界限制多能干细胞在二维平面上的生长，并向神经方向诱导分化，最终形成具有厚度在500-1500μm和特定形状的大脑类器官模型，可实现高通量、一步法构建形态均一的类器官，并可原位观察类器官生长、发育全过程；
(2)一步法：无需转移培养位置，即可完成大脑类器官全流程的培养，简化培养步骤
(3)本发明中的大脑类器官，可以在孔板中进行原位分析，不会破坏类器官结构，对现有的用于药物开发领域的生物分析和成像仪器(如高内涵仪器)均有良好的兼容性。
(4)本发明的大脑类器官模型与已有的培养大脑类器官的方法对比具有明显的差异及优势，具体表现在：经典的大脑类器官培养方法均需使悬浮的单个细胞自发聚集成拟胚体小球，在该阶段难以控制最终成球的细胞量；形成的拟胚体需要多次转移至不同孔板中；大脑类器官为悬浮动态培养，难以实时观察类器官培养状态等，都是影响大脑类器官标准化的障碍。而本发明直接利用单层贴壁培养的多能干细胞，通过边界限制其生长范围，创造了一种贴壁生长的大脑类器官模型及其制备方法，制备方法的相关参数已被确定，类器官形成位置、形态、大小都具有高度的均一性，且通量高，易于原位观察和实时成像。本发明创造的模型及制作方法对比传统的大脑类器官培养方法存在显著的差别，且具有无可 比拟的优势，解决了大脑类器官培养复杂、通量低、难以原位观察的难题。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1为本发明实施例提供的大脑类器官的培养芯片的制作示意图；
图2a为图案化芯片；图2b为图案化大脑类器官明场图片；图2c为培养至第25天时图案化与传统方法培养的大脑类器官面积比较的结果，图2d为培养至第25天时图案化与传统方法培养的大脑类器官面积的变异系数比较的结果；
图3为明场条件下图案化大脑类器官发育情况的实时监测结果；
图4为免疫荧光以及冰冻切片方法在蛋白水平检测图案化大脑类器官的发育情况，及加入不同浓度Aβ42O处理后的MAP2基因表达，比例尺为100μm。
图5为明场条件下不同尺寸图案化大脑类器官发育情况的监测结果，比例尺为100μm。
图6为加入5μM的Aβ42O处理后，神经元死亡情况，染色方法为TUNEL染色，比例尺为50μm。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
需要理解的是，术语“长度”、“宽度”、“上”、下”、“前”、“后”、“第一”、“第二”、“竖直”、“水平”、“顶”、“底”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
此外，在本申请的描述中，“多个”、“若干个”的含义是两个或两个以上，除非另有明确具体的限定。
本申请的技术方案总体思路如下：
根据本发明的一种典型的实施方式，提供大脑类器官模型的制备方法，所述方法包括：
步骤S1、获得具有边界限制的单层贴壁生长的多能干细胞；
所述获得具有边界限制的单层贴壁生长的多能干细胞中的边界限制的方法包括分子印章法、光刻法、穿孔薄膜法和差异性粘附法中的一种。
作为一种具体的实施方式，采用穿孔薄膜法，具体操作如下：
步骤S101、获得具有多个穿孔的薄膜；
其中，所述步骤S101具体包括：
步骤S1011、获得具有微柱阵列的阳模；所述阳膜的所述微柱阵列高度为30μm～100μm。若高度过低则薄膜过薄，难以操作；若高度过高，则不易穿孔。
步骤S1012、将PDMS倒入所述阳模上，真空干燥并抽真空，并在所述PDMS上覆盖一层PMMA，后用两个玻璃片夹紧固定，烘干；
步骤S1013、将凝固的PDMS层取出，获得具有多个穿孔的薄膜。
所述薄膜的材料具体可以为聚二甲基硅氧烷。
所述薄膜外形尺寸与所述细胞培养板相匹配；
所述薄膜的每个所述穿孔的孔形状包括圆形、椭圆形、半圆形、扇形、三角形、四边形、五边形、六边形和任意多边形中的一种；
所述薄膜中穿孔的排列方式包括：正方形顶点排列，正六边形排列、线性排列和任意排列方式中的一种。
所述薄膜的每个所述穿孔面积为0.005～3.5mm2，所述薄膜的相邻两个穿孔的间距为0.5～245mm。若穿孔面积小于0.005mm2，存在换液时易丢失的缺点，若面积多大，则难以形成大脑类器官；若所述薄膜的相邻两个穿孔的间距小于0.5mm，存在类器官融合的缺点，若间距过大，则无法适配现有细胞培养系统；多个所述穿孔可规则排布，也可不规则排布。
所述细胞培养板包括384孔板、96孔板、48孔板、24孔板、12孔板、6孔板中的一种。
当采用细胞培养皿时，所述细胞培养皿包括3.5cm培养皿、6cm培养皿和10cm培养皿中的一种。
步骤S102、将所述薄膜置于细胞培养板底部，并向所述细胞培养板中加入细胞粘附材料进行包被；
所述细胞黏附材料包括Matrigel和Vitronectin中的至少一种；所述细胞黏附材料的浓度为0.1～1mg/mL。若浓度过低，则存在细胞难以贴附的缺点；若浓度过高，则难以去除穿孔薄膜，细胞图案化效果不佳。
步骤S103、向所述包被后细胞培养板内接种多功能干细胞，待细胞贴壁后，将所述薄膜移走，获得具有边界限制的单层贴壁生长的多能干细胞。
上述为穿孔薄膜法的具体步骤，所述获得具有边界限制的单层贴壁生长的多能干细胞中的边界限制的方法包括分子印章法、光刻法、穿孔薄膜法和差异性粘附法中的一种。也可采用其他方法获得具有边界限制的单层贴壁生长的多能干细胞，只需控制所述具有边界限制的单层贴壁生长的多能干细胞的生长面积为0.005～3.5mm2，相邻两个所述具有边界限制的单层贴壁生长的多能干细胞区域的间距为0.5～245mm。生长面积过小存在换液时易丢失的缺点，若多大，则难以形成大脑类器官；若间距小于0.5mm，存在类器官融合的缺点，若间距过大，则无法适配现有细胞培养系统；多个所述穿孔可规则排布，也可不规则排布。所述边界限制的几何形状包括圆形、椭圆形、半圆形、扇形、三角形、四边形、五边形、六边形和任意多边形中的一种；述边界限制的多能干细胞排列方式包括：正方形顶点排列，正六边形排列、线性排列和任意排列方式中的一种。
所述人多功能干细胞(人胚胎干细胞或人诱导多能干细胞)的接种密度为1×105～1×106(个/cm2)。所述多能干细胞包括人胚胎干细胞和人诱导多能干细胞中的一种，接种密度为105-106个细胞每平方厘米。若接种密度过低，则存在短时间内细胞难以融合，不易成为具有一定厚度的大脑类器官的缺点；若密度过高，则影响细胞开始分化时的状态，培养过程中死细胞过多。
步骤S2、向所述具有边界限制的单层贴壁生长的多能干细胞中加入EB形成培养基维持培养4～6天，采用神经诱导培养基培养2～6天，后吸弃所述神经诱导培养基培养基，加入预冷的Matrigel进行包被，固化后，依次更换神经分化培养基培养3～5天、诱导成熟培养基培养10～30天，获得大脑类器官模型。
所述步骤S2中，
加入EB形成培养基维持培养4～6天，是为了促进细胞增殖并堆叠呈实心圆拱形；
后采用神经诱导培养基培养2～6天，是为了诱导细胞向神经外胚层分化同时继续增殖；
作为一种具体的实施方式，所述EB形成培养基的基础成分为NuwacellTM hiPSC/hESC培养基-ncTarget，另外添加ROCK inhibitor Y27632和bFGF；所述的ROCK inhibitor Y27632的母液浓度为10mM；所述的ROCK inhibitor Y27632的母液与ncTarget的体积比为1:1000；所述的bFGF的终浓度为2～6ng/mL，优选4ng/ml。
作为一种具体的实施方式，所述神经诱导培养基的基础成分为DMEM/F12，另外需添加占总体积1％NEAA(Non Essential Amino Acid，100×)，占总体积1％GlutaMAX(100×)(可购买于GIBCO/Invitrogen货号35050061)，占总体积1％N2(100×)(N-2添加剂)， 0.5～2μg/mL heparin(优选1μg/ml)，占总体积1％penicillin-streptomycin(100×)。
作为一种具体的实施方式，所述的神经分化培养基的基础成分为DMEM/F12和Neurobasal medium，其体积比为1:1，另外需添加占总体积1％B27without VitaminA(50×)，占总体积0.5％N2(100×)，占总体积0.5％NEAA(100×)，占总体积1％GlutaMAX(100×)，占总体积1％penicillin-streptomycin(100×)，2～5μg/ml beta-mercaptoethanol，优选3.5μg/ml，1～4μg/ml insulin solution，优选2.5μg/ml。
作为一种具体的实施方式，所述的诱导成熟培养基的基础成分为DMEM/F12和Neurobasal medium，其体积比为1:1，另外需添加占总体积1％B27(50×)，占总体积0.5％N2(100×)，占总体积0.5％NEAA(100×)，占总体积1％GlutaMAX(100×)，占总体积1％penicillin-streptomycin(100×)，2～5μg/ml beta-mercaptoethanol，优选3.5μg/ml，1～4μg/ml insulin solution，优选2.5μg/ml。
上述技术方案的创新之处在于：(1)此前不存在贴壁培养的类器官，从单层贴壁细胞开始培养的方案不是显而易见的：单层细胞会均匀接触到分化因子，从而难以由于因子的浓度梯度、细胞间相互作用等产生具有异质性自组织的结构，因此大脑类器官培养都是从EB开始的，我们通过对单层培养细胞进行形状的限制，从而使细胞出现三维层面上的堆积，加上细胞外基质的支撑，从而发育成大脑类器官，拓宽了大脑类器官的生成思路。(2)一步法：无需转移培养位置，即可完成大脑类器官全流程的培养，简化培养步骤。(3)传统大脑类器官培养方法在生长后期阶段，直径达到3～4mm。然而，大脑类器官内部含有大量细胞缺氧坏死去。本方法构建的图案化大脑了器官能够极大减少内部细胞坏死。
具体通过图案化技术在细胞培养器皿底部形成具有特定图案的细胞特异性黏附区域，在此图案化基底上可以接种多能干细胞，待细胞贴壁后，将穿孔薄膜移除，获得具有特定边缘形状的细胞聚集体，在此图案化基底上诱导干细胞向大脑类器官分化，最终形成具有厚度在500～1500μm和特定边缘形状的大脑类器官模型，均一性高且高通量，适用于基于细胞培养孔板的一系列成像及分析仪器。
根据本发明另一种典型实施方式，提供了采用所述的方法获得的大脑类器官模型。作为本发明实施例一种具体的实施方式，获得的大脑类器官是球缺形，即一个球被平面截下一部分后的形状，这是不同于大脑类器官的表型；外周随机分布神经花环状结构，偶然情况下部分大脑类器官周围会出现空泡状结构。
根据本发明另一种典型实施方式，提供了所述的大脑类器官模型在神经毒性分析中的应用。
所述大脑类器官模型制备方法包括穿孔薄膜的获得，图案化芯片制备，将多能干细胞 接种在图案化基底上，向神经外胚层方向分化，最终形成粘附于培养皿底部同时具有一定厚度和特定边缘形状的球缺形大脑类器官，其包含的细胞类型包括干细胞来源的神经干细胞、神经元、星形胶质细胞、小胶质细胞；所述大脑类器官可以应用于研究大脑发育、疾病、药物筛选、药物神经毒性评价等。与传统大脑类器官培养方法相比，本发明提供了一种高通量、高均一性、原位培养的大脑类器官模型，为相关大脑类器官研究提供了一个创新研究工具。
下面将结合实施例和附图对本申请的一种大脑类器官模型及其制备方法与应用进行详细说明。
实施例1一种大脑类器官模型的培养方法
一、边界限制多能干细胞的制备方法
使用软光刻技术制备SU-8模板，此模板为高通量微柱阵列，微柱直径为500微米，高度为50微米，微柱间距为1000微米；
如图1所示，将PDMS预聚物与固化剂按10:1比例混合后倾倒到SU-8模板上，抽真空去除气泡后，在上面覆盖一层0.2mm的PMMA板，用两个盖玻片夹持并用台虎钳固定，置于80℃烘箱聚合处理120min，取出待恢复至室温后将穿孔PDMS薄膜从SU-8模板上剥离，用直径10mm打孔器取下适合大小的薄膜，在48孔板中加入1mL 70％乙醇，将薄膜置于板底，吸弃多余乙醇，80℃烘箱干燥，得到如图2a所示图案化芯片；
将图案化芯片置于紫外灯下灭菌处理60min，每孔加入500μL 1×DPBS润洗芯片，移除DPBS后加入0.2mg/mL Matrigel:DMEM/F12，37℃孵育1h备用；
将融合度为70％-80％的hiPSCs用Accutase消化成单个细胞，用nctarget重悬，吸弃2％Matrigel:DMEM/F12，每孔加入1.5×105个细胞，待细胞贴壁后用镊子小心移除PDMS薄膜，得到具有边界限制多能干细胞。
所述多能干细胞的边界限制形状为圆形；
所述多能干细胞的生长面积(即所述薄膜的每个所述穿孔面积)为0.2mm2；
所述多能干细胞的生长区域间距(即所述薄膜的相邻两个穿孔的间距)为1.5mm；
所述多能干细胞的生长区域排列方式为正方形顶点排列；
所述大脑类器官的培养体系为48孔板。
二、图案化大脑类器官的培养方法
换用EB形成培养基培养6天，培养后效果如图2(右)；
待细胞增厚后，换用神经诱导培养基培养5天；
吸弃原有培养基，1×DPBS小心润洗两次，用预冷的Matrigel对图案化细胞包被，37℃ 二氧化碳培养箱中固化37min后，加入神经分化培养基培养3天；
更换诱导成熟培养基持续培养，一段时间后，对形成的图案化大脑类器官进行观察；
明场跟踪上述方法诱导的图案化大脑类器官的发育状况，结果如图3所示。
实施例2
该实施例中，多能干细胞的边界限制形状为圆形；所述多能干细胞的生长面积为0.008mm2；所述多能干细胞的生长区域间距为1mm；所述多能干细胞的生长区域排列方式为正方形顶点排列；所述大脑类器官的培养体系为48孔板；其他均同实施例1。
实施例3
该实施例中，多能干细胞的边界限制形状为圆形；所述多能干细胞的生长面积为0.03mm2；所述多能干细胞的生长区域间距为1.2mm；所述多能干细胞的生长区域排列方式为正方形顶点排列；所述大脑类器官的培养体系为48孔板；其他均同实施例1。
实施例4
该实施例中，多能干细胞的边界限制形状为圆形；所述多能干细胞的生长面积为0.8mm2；所述多能干细胞的生长区域间距为2mm；所述多能干细胞的生长区域排列方式为正方形顶点排列；所述大脑类器官的培养体系为48孔板；其他均同实施例1。
实施例5
该实施例中，多能干细胞的边界限制形状为正方形；所述多能干细胞的生长面积为1mm2；所述多能干细胞的生长区域间距为2mm；所述多能干细胞的生长区域排列方式为正方形顶点排列；所述大脑类器官的培养体系为48孔板；其他均同实施例1。
实施例6
该实施例中，多能干细胞的边界限制形状为正三角形；所述多能干细胞的生长面积为0.43mm2；所述多能干细胞的生长区域间距为2mm；所述多能干细胞的生长区域排列方式为正方形顶点排列；所述大脑类器官的培养体系为48孔板；其他均同实施例1。
实施例7
该实施例中，多能干细胞的边界限制形状为十字形；所述多能干细胞的生长面积为0.2 mm2；所述多能干细胞的生长区域间距为1mm；所述多能干细胞的生长区域排列方式为正方形顶点排列；所述大脑类器官的培养体系为48孔板；其他均同实施例1。
实施例8
该实施例中，多能干细胞的边界限制形状为圆形；所述多能干细胞的生长面积为0.2mm2；所述多能干细胞的生长区域间距为1mm；所述大脑类器官的培养体系为24孔板；其他均同实施例1。
实施例9
该实施例中，多能干细胞的边界限制形状为圆形；所述多能干细胞的生长面积为0.2mm2；所述多能干细胞的生长区域间距为1.5mm；所述穿孔排列方式为正六边形排列；所述大脑类器官的培养体系为24孔板；其他均同实施例1。
实施例10
该实施例中，图案化大脑类器官芯片的细胞粘附物质为Vitronectin，其他均同实施例1。
对比例1
该对比例中多能干细胞的边界限制面积为0.002mm2，其他均同实施例1。
对比例2
该对比例中多能干细胞的边界限制面积为5mm2，其他均同实施例1。
对比例3
该对比例中多能干细胞的生长区域间距为0.1mm，其他均同实施例1。
对比例4
该对比例中多能干细胞的生长区域间距为0.05mm，其他均同实施例1。
对比例5
该对比例中大脑类器官开始分化的细胞密度为5×103/cm2，其他均同实施例1。
对比例6
该对比例中大脑类器官开始分化的细胞密度为5×107/cm2，其他均同实施例1。
对比例7
该对比例中多能干细胞的细胞粘附物质为10mg/mL Matrigel，其他均同实施例1。
对比例8
该对比例中多能干细胞的细胞粘附物质为5mg/mL Matrigel，其他均同实施例1。
对比例9
该对比例中多能干细胞的细胞粘附物质为0.01mg/mL Matrigel，其他均同实施例1。
实验例1
对上述实施例1-10和对比例1-9的图案化大脑类器官培养效果进行统计，如表1所示，其中面积的标准差变异系数的计算方法为：变异系数C·V＝(标准偏差SD/平均值Mean)×100％；
表1

由表1的数据可知：
对比例1中，多能干细胞的边界限制面积为0.002mm2，小于本发明实施例0.005～3.5mm2的范围，存在类器官贴附不紧密，换液过程中易丢失的缺点；
对比例2中，多能干细胞的边界限制面积为5mm2，大于本发明实施例0.005～3.5mm2的范围，存在图案化大脑类器官难以培养成型的缺点；
对比例3中，多能干细胞的生长区域间距为0.1mm，小于本发明实施例0.5～245mm的范围，在类器官生长增大过程中会造成融合，存在难以形成独立类器官的缺点；
对比例4中，多能干细胞的生长区域间距为0.05mm，小于本发明实施例0.5～245mm 的范围，在类器官生长增大过程中会造成融合，存在难以形成独立类器官的缺点；
对比例5中，大脑类器官开始分化的细胞密度为5×103/cm2，小于本发明实施例105～106的范围，存在初始细胞密度过低，难以形成多种细胞类型的类器官的缺点；
对比例6中，大脑类器官开始分化的细胞密度为5×107/cm，大于本发明实施例105～106的范围，存在初始细胞密度过高，影响后续分化状态的缺点；
对比例7中，多能干细胞的细胞粘附物质为10mg/mL Matrigel，大于本发明实施例0.1～1mg/mL的范围，存在诱导细胞向其他方向提前分化的缺点；
对比例8中，多能干细胞的细胞粘附物质为5mg/mL Matrigel，大于本发明实施例0.1～1mg/mL的范围，存在诱导细胞向其他方向提前分化的缺点；
对比例9中，多能干细胞的细胞粘附物质为0.01mg/mL Matrigel，小于本发明实施例0.1～1mg/mL的范围，存在细胞外粘附物质浓度过低，细胞难以贴附的缺点；
本发明实施例1-实施例10中，形成具有厚度在500-1500μm和特定边缘形状的大脑类器官模型，均一性高。
将实施例1培养至第25天时图案化大脑类器官与传统方法培养的大脑类器官面积和变异系数进行比较；由图2c和图2d可知，培养至后期的图案化大脑类器官的大小与传统方法培养的大脑类器官的大小基本一致，但本发明实施例的大脑类器官均一性上好于传统方法培养的大脑类器官。
综上可知，本发明利用对单层培养的多能干细胞进行边界限制，生成了贴壁培养的球缺状的新型大脑类器官模型，该模型具有神经元、神经干细胞、星形胶质细胞等大脑相关细胞类型，可实现高通量、一步法构建形态均一的大脑类器官，避免了类器官培养过程中由方法和操作导致的融合及污染的风险，并可原位观察类器官生长、发育全过程，用于大脑类器官的应用研究。
实验例2人源大脑类器官标志物检测
1、使用免疫荧光检测大脑类器官中不同细胞类型标志物：在培养至25天时对图案化大脑类器官固定并切片。NEXTIN和SOX2共染确定神经干细胞分化情况，TUJ和MAP2鉴定神经元的分化，PAX6鉴定图案化大脑类器官中前脑相关标记的表达。
2、实施例1图案化基底上的Aβ42寡聚体神经毒性评价体系的构建与表征：在第25天时，分别使用Control、0μM、1μM、5μM的Aβ42寡聚体(Aβ42O)对大脑类器官类器官处理72小时后进行QPCR检测神经元相关标志物的表达。结果如图4，表明本发明实施例成功 获得人源大脑类器官并构建了Aβ42寡聚体神经毒性评价体系。
3、实施例1不同直径的图案化基底，包括直径100、200、500μm的图案化大脑类器官。
4、实施例1图案化基底上的Aβ42寡聚体神经毒性评价体系的构建与表征：在第25天时，分别使用Control、5μM的Aβ42寡聚体(Aβ42O)对大脑类器官类器官处理72小时后进行TUNEL染色检测神经元凋亡情况。结果如图6，表明本发明实施例成功获得人源大脑类器官并构建了Aβ42寡聚体神经毒性评价体系。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。

Claims (15)

  1. 一种大脑类器官模型的制备方法，其特征在于，所述方法包括：
    获得具有边界限制的单层贴壁生长的多能干细胞；
    向所述具有边界限制的单层贴壁生长的多能干细胞中加入EB形成培养基维持培养4～6天，采用神经诱导培养基培养2～6天，后吸弃所述神经诱导培养基培养基，加入预冷的Matrigel进行包被，固化后，依次更换神经分化培养基培养3～5天、诱导成熟培养基培养10～30天，获得大脑类器官模型。
  2. 根据权利要求1所述的一种大脑类器官模型的制备方法，其特征在于，所述获得具有边界限制的单层贴壁生长的多能干细胞中的边界限制的方法包括分子印章法、光刻法、穿孔薄膜法和差异性粘附法中的一种。
  3. 根据权利要求1所述的一种大脑类器官模型的制备方法，其特征在于，所述获得具有边界限制的单层贴壁生长的多能干细胞，具体包括：
    获得具有多个穿孔的薄膜；
    将所述薄膜置于细胞培养板底部，并向所述细胞培养板中加入细胞粘附材料进行包被；
    向所述包被后细胞培养板内接种多功能干细胞，待细胞贴壁后，将所述薄膜移走，获得具有边界限制的单层贴壁生长的多能干细胞。
  4. 根据权利要求3所述的一种大脑类器官模型的制备方法，其特征在于，所述薄膜外形尺寸与所述细胞培养板相匹配；所述薄膜的每个所述穿孔的孔形状包括圆形、椭圆形、半圆形、扇形、三角形、四边形、五边形、六边形和任意多边形中的一种；所述薄膜中穿孔的排列方式包括：正方形顶点排列，正六边形排列、线性排列和任意排列方式中的一种。
  5. 根据权利要求3所述的一种大脑类器官模型的制备方法，其特征在于，所述细胞黏附材料包括Matrigel和Vitronectin中的至少一种；所述细胞黏附材料的浓度为0.1～1mg/mL。
  6. 根据权利要求1所述的一种大脑类器官模型的制备方法，其特征在于，所述多能干细胞包括人胚胎干细胞或人诱导多能干细胞中的一种。
  7. 根据权利要求1所述的一种大脑类器官模型的制备方法，其特征在于，所述具有边界限制的单层贴壁生长的多能干细胞的生长面积为0.005～3.5mm2，相邻两个所述具有边界限制的单层贴壁生长的多能干细胞区域的间距为0.5～245mm。
  8. 一种利用权利要求1～7任意一项所述的方法获得的大脑类器官模型。
  9. 根据权利要求8所述的大脑类器官模型，其特征在于，所述大脑类器官模型中大脑类器官形状为球缺形，即一个球被平面截下一部分后的形状；外周随机分布神经花环状结构。
  10. 一种权利要求9所述的大脑类器官模型在神经机制研究、神经疾病模型、神经药 物开发及神经毒性分析中的应用。
  11. 根据权利要求1所述的一种大脑类器官模型的制备方法，其特征在于，所述诱导成熟培养基培养方式为倾斜角≤25°的翘板摇床动态培养。
  12. 根据权利要求3所述的一种大脑类器官模型的制备方法，其特征在于，所述细胞培养板包括384孔板、96孔板、48孔板、24孔板、12孔板、6孔板中的一种。
  13. 根据权利要求3所述的一种大脑类器官模型的制备方法，其特征在于，所述细胞培养板底部厚度为0.1-1.3mm。
  14. 根据权利要求1所述的一种大脑类器官模型的制备方法，其特征在于，所述获得具有边界限制的单层贴壁生长的多能干细胞，具体包括：
    获得具有多个穿孔的薄膜；
    将所述薄膜置于细胞培养皿底部，并向所述细胞培养皿中加入细胞粘附材料进行包被；
    向所述包被后细胞培养皿内接种多功能干细胞，待细胞贴壁后，将所述薄膜移走，获得具有边界限制的单层贴壁生长的多能干细胞。
  15. 根据权利要求14所述的一种大脑类器官模型的制备方法，其特征在于，所述细胞培养皿包括3.5cm培养皿、6cm培养皿和10cm培养皿中的一种。
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