CN114657127A - Brain organoid model and preparation method and application thereof - Google Patents
Brain organoid model and preparation method and application thereof Download PDFInfo
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- CN114657127A CN114657127A CN202210185202.2A CN202210185202A CN114657127A CN 114657127 A CN114657127 A CN 114657127A CN 202210185202 A CN202210185202 A CN 202210185202A CN 114657127 A CN114657127 A CN 114657127A
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Abstract
The invention discloses a brain organoid model and a preparation method and application thereof, wherein the method comprises the following steps: obtaining pluripotent stem cells with boundary limit and single-layer adherent growth, and adding EB (Epstein-Barr) to form a culture medium to maintain and culture for 4-6 days; then adopting a nerve induction culture medium to culture for 2-6 days; and (3) absorbing and removing the culture medium, coating the culture medium with pre-cooled Matrigel, solidifying, and sequentially replacing a neural differentiation culture medium for culturing for 3-5 days and an induction maturation culture medium for culturing for 10-30 days to obtain the brain organoid model. The invention realizes the generation of the brain organoid model from monolayer cell culture for the first time, can realize the construction of organoids with uniform shape by a high-flux and one-step method, and can observe the whole growth and development process of the organoids in situ.
Description
Technical Field
The invention relates to the technical field of stem cells, organoids, organoid chips and tissue engineering, in particular to a brain organoid model and a preparation method and application thereof.
Background
The brain is the most complex organ of human whole body structure and function, and understanding human brain development and disease is one of the greatest challenges in life sciences. However, the difficulty in obtaining human brain tissue has severely hampered our step in breaking the secret of the human brain. Researchers have long used cell culture models and animal models to study adult human brain development and disease. These studies lay the foundation for our current understanding of brain development and function. Nevertheless, our understanding of the human brain is limited to simple cell-cell interactions and features common to humans and vertebrates. Therefore, in order to further research the development mechanism of human brain, research the pathogenesis and treatment method of brain-related diseases, and establish an in vitro brain research model highly related to human body, have important significance. Brain organoids are an emerging brain research model with key features of the human brain, such as various brain-specific cell types, apical-basal polarity, neural stem cell division and neuronal migration patterns. Compared with the traditional animal model, the brain organoid model has no species difference and has a structure and a cell type which are highly related to the human body; compared with a two-dimensional culture model, the method has the advantages of similar cell microenvironment to the in vivo, multiple brain cell populations, capability of simulating nerve electrical signals in the brain and the like. Therefore, the in vitro brain organoid construction provides an effective model system for researching human brain development and diseases.
After the brain organoid is cultured for the first time by madelin Lancaster in 2013, the mainstream brain organoid culture schemes are all the culture schemes or variants thereof, and mainly comprise four stages: embryoid body formation, neuroectodermal induction, neuroepithelial differentiation, and brain organoid maturation. Because the monolayer adherent cells can uniformly contact with the differentiation factors in the growth process, the heterogeneous self-organized structure is difficult to generate due to the concentration gradient of the factors, the interaction between cells and the like, and therefore, in the process of culturing the brain organoid, the stem cells need to be firstly formed into embryoid bodies globules on a low-adhesion U-shaped bottom orifice plate or by adopting a pendant drop method, then the embryoid bodies subjected to nerve induction are coated with Matrigel, and then the embryoid bodies are transferred to a low-adhesion culture plate or a bioreactor for dynamic culture. However, during organoid transfer and suspension culture, organoids are subject to external stimuli from human manipulation, as well as positional uncertainty, are highly susceptible to contamination or fusion, and are difficult to observe positionally. The above limitations result in organoid culture processes that are complex, highly diverse, low-throughput and not easily monitored in real-time. In the existing brain organoid culture methods, cell balling is required to start.
Therefore, in order to better solve the problems of the existing brain organoid culture methods, it is necessary to develop an in-situ high-throughput and high-homogeneity brain organoid model for the application research of brain organoids.
Disclosure of Invention
The invention aims to provide a brain organoid model, a preparation method and application thereof, which can realize high-throughput and one-step construction of a brain organoid with uniform morphology and can observe the whole growth and development process of the organoid in situ.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method of making a brain organoid model, the method comprising:
obtaining a unilamellar adherently growing pluripotent stem cell with boundary limitations;
and adding EB (Epstein-Barr) into the unilamellar adherent growth pluripotent stem cells with the boundary limitation to form a culture medium for maintaining and culturing for 4-6 days, adopting a neural induction culture medium for culturing for 2-6 days, then removing the neural induction culture medium, adding pre-cooled Matrigel for coating, solidifying, and sequentially replacing a neural differentiation culture medium for culturing for 3-5 days and an induction maturation culture medium for culturing for 10-30 days to obtain the brain organoid model.
Further, the method of obtaining boundary confinement in a monolayer adherently grown pluripotent stem cell with boundary confinement comprises one of a molecular imprinting method, a photolithography method, a perforated film method, and a differential adhesion method.
Further, the obtaining of the unilamellar adherently grown pluripotent stem cells with boundary limitation specifically comprises:
obtaining a film having a plurality of perforations;
placing the film at the bottom of a cell culture plate, and adding a cell adhesion material into the cell culture plate for coating;
inoculating multifunctional stem cells into the coated cell culture plate, removing the film after the cells adhere to the wall, and obtaining the cell culture plate
A unilamellar adherently growing pluripotent stem cell with boundary limitations.
Further, the film physical dimensions match the cell culture plate; the aperture shape of each of the perforations of the film comprises one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrangle, a pentagon, a hexagon, and any polygon; the arrangement of the perforations in the film comprises: square vertex arrangement, regular hexagon arrangement, linear arrangement and random arrangement.
Further, 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.
Further, the pluripotent stem cells comprise one of human embryonic stem cells or human induced pluripotent stem cells;
further, the growth area of the unilamellar adherent growth pluripotent stem cells with the boundary limitation is 0.005-3.5 mm2And the distance between two adjacent unilamellar adherent growth pluripotent stem cell areas with boundary limitation is 0.5-245 mm.
Further, the boundary-restricted pluripotent stem cells have a density of 1X 10 at the start of addition to the EB forming medium5～1×106(pieces/cm)2)。
In the technical scheme, the shape of the generated brain organoid is in a segment shape, namely a shape of a sphere which is cut by a plane, and a nerval ring-shaped structure is randomly distributed on the periphery; brain organoids include, but are not limited to, neurons, neural stem cells, astrocytes, microglial brain-associated cell types.
In a second aspect of the invention, there is provided a brain organoid model obtained using the method.
In a third aspect of the invention, the application of the brain organoid model in neural mechanism research, neural disease models, neurodrug development and neurotoxicity analysis is provided.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
(1) according to the brain organoid model and the preparation method and application thereof provided by the invention, the growth of multipotential stem cells on a two-dimensional plane is limited by a boundary, and induced differentiation is carried out towards the nerve direction, and the brain organoid model with the thickness of 500-;
(2) a one-step method: the whole process of culturing the brain organoid can be completed without transferring the culture position, and the culturing steps are simplified
(3) The brain organoid of the present invention can be analyzed in situ in the well plate without destroying the organoid structure, and has good compatibility with the existing bioanalysis and imaging instruments (such as high content instruments) used in the field of drug development.
(4) Compared with the existing method for culturing the brain organoid, the brain organoid model of the invention has obvious differences and advantages, which are specifically shown in the following steps: the classical brain organoid culture methods all require the spontaneous aggregation of suspended single cells into embryoid body globules, and the amount of cells which finally form globules is difficult to control at this stage; the formed embryoid body needs to be transferred to different pore plates for many times; the brain organoid is in suspension dynamic culture, the culture state of the organoid is difficult to observe in real time, and the like, and the brain organoid is an obstacle influencing the standardization of the brain organoid. The invention directly utilizes the pluripotent stem cells cultured by monolayer adherent culture, limits the growth range of the pluripotent stem cells through the boundary, creates an adherent growth brain organoid model and a preparation method thereof, the relevant parameters of the preparation method are determined, the organoid formation position, the morphology and the size have high uniformity, the flux is high, and the in-situ observation is easy. Compared with the traditional brain organoid culture method, the model and the manufacturing method provided by the invention have obvious difference, have incomparable advantages and solve the problems of complex brain organoid culture, low flux and difficulty in-situ observation.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a schematic diagram illustrating a method for manufacturing a culture chip for a brain organoid according to an embodiment of the present invention;
FIG. 2a is a patterned chip; FIG. 2b is a photograph of a patterned brain organoid bright field; FIG. 2c shows the results of comparing the area of patterned and conventionally cultured brain organoids by day 25, and FIG. 2d shows the results of comparing the coefficient of variation of the area of patterned and conventionally cultured brain organoids by day 25;
FIG. 3 is a real-time monitoring result of the development of patterned brain organoids under bright field conditions;
FIG. 4 shows immunofluorescence and cryosectioning assays for patterned brain organoid development at the protein level and different Abeta concentrations42MAP2 gene expression after O treatment, at a scale bar of 100 μm.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are provided to illustrate the invention, and not to limit the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "first," "second," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, as used herein, refer to an orientation or positional relationship indicated in the drawings that is solely for the purpose of facilitating the description and simplifying the description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be considered as limiting the present application.
In addition, in the description of the present application, "a plurality" or "a plurality" means two or more unless specifically defined otherwise.
The technical scheme of the application has the following general idea:
according to an exemplary embodiment of the present invention, a method for preparing a brain organoid model is provided, the method comprising:
step S1, obtaining the unilamellar adherent growth pluripotent stem cells with boundary limitation;
the method for obtaining boundary confinement in a monolayer adherently grown pluripotent stem cell with boundary confinement comprises one of a molecular imprinting method, a photolithography method, a perforated film method and a differential adhesion method.
As a specific implementation mode, a perforated film method is adopted, and the specific operation is as follows:
step S101, obtaining a film with a plurality of perforations;
wherein, the step S101 specifically includes:
step S1011, obtaining a male die with a micropillar array; the height of the micro-column array of the anode membrane is 30-100 mu m. If the height is too low, the film is too thin and is difficult to operate; if the height is too high, the hole is not easily punched.
Step S1012, pouring PDMS (polydimethylsiloxane) on the male mold, performing vacuum drying and vacuumizing, covering a layer of PMMA on the PDMS, clamping and fixing by two glass sheets, and drying;
step S1013, taking out the solidified PDMS layer to obtain a film having a plurality of perforations.
The material of the film may specifically be polydimethylsiloxane.
The overall dimension of the film is matched with that of the cell culture plate;
the shape of each perforated hole of the film comprises one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrilateral, a pentagon, a hexagon and an arbitrary polygon;
the arrangement of the perforations in the film comprises: square vertex arrangement, regular hexagon arrangement, linear arrangement and random arrangement.
Each perforation area of the film is 0.005-3.5 mm2And the distance between two adjacent through holes of the film is 0.5-245 mm. If the area of the through hole is less than 0.005mm2The defect that the liquid is easy to lose when the liquid is changed exists, and if the area is large, the brain organoid is difficult to form; if the distance between two adjacent perforations of the film is less than 0.5mm, the defect of organoid fusion exists, and if the distance is too large, the film cannot be adapted to the existing cell culture system; the plurality of through holes may be regularly arranged or irregularly arranged.
The cell culture plate comprises one of a 384-well plate, a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate, a 3.5-cm culture dish, a 6-cm culture dish, and a 10-cm culture dish.
Step S102, placing the film at the bottom of a cell culture plate, and adding a cell adhesion material into the cell culture plate for coating;
the cell adhesion material comprises 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 the cells are difficult to adhere; if the concentration is too high, it is difficult to remove the perforated film, and the cell patterning effect is not good.
And S103, inoculating multifunctional stem cells into the coated cell culture plate, and removing the film after the cells adhere to the wall to obtain the unilamellar adherent growth multifunctional stem cells with boundary limitation.
The above is a specific embodiment of the perforated film methodA method of obtaining boundary confinement in a monolayer adherently grown pluripotent stem cell with boundary confinement includes one of a molecular imprinting method, a photolithography method, a perforated film method, and a differential adhesion method. The pluripotent stem cells with boundary limit and single-layer adherent growth can also be obtained by other methods, and the growth area of the pluripotent stem cells with boundary limit and single-layer adherent growth only needs to be controlled to be 0.005-3.5 mm2And the distance between two adjacent unilamellar adherent growth pluripotent stem cell areas with the edge limitation system is 0.5-245 mm. The growth area is too small, so that the liquid is easy to lose when being changed, and if the growth area is too large, the brain organoid is difficult to form; if the spacing is less than 0.5mm, the defect of organoid fusion exists, and if the spacing is too large, the cell culture system cannot be adapted to the existing cell culture system; the plurality of through holes may be regularly arranged or irregularly arranged. The geometric shape of the boundary restriction comprises one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrangle, a pentagon, a hexagon and an arbitrary polygon; the boundary-limited pluripotent stem cell arrangement comprises: square vertex arrangement, regular hexagon arrangement, linear arrangement and random arrangement.
The seeding density of the human pluripotent stem cells (human embryonic stem cells or human induced pluripotent stem cells) is 1 multiplied by 105～1×106(pieces/cm)2). The pluripotent stem cells comprise one of human embryonic stem cells and human induced pluripotent stem cells, and the seeding density is 105-106Each cell per square centimeter. If the inoculation density is too low, cells are difficult to fuse in a short time, and the cells are not easy to become a brain organoid with a certain thickness; if the density is too high, the state of the cells at the time of starting differentiation is affected, and the number of dead cells during culture is too large.
Step S2, adding EB into the unilamellar adherent growth pluripotent stem cells with the boundary limitation to form a culture medium, maintaining the culture for 4-6 days, adopting a neural induction culture medium to culture for 2-6 days, then sucking and removing the neural induction culture medium, adding pre-cooled Matrigel to coat, and after solidification, sequentially replacing a neural differentiation culture medium to culture for 3-5 days and inducing a mature culture medium to culture for 10-30 days to obtain the brain organoid model.
In the step S2, in the above step,
EB is added to form a culture medium for maintaining culture for 4-6 days, so as to promote cell proliferation and stack the cells into a solid circular arch shape;
then, a neural induction culture medium is adopted for culturing for 2-6 days in order to induce the cells to differentiate towards neuroectoderm and continue to proliferate;
as a specific embodiment, the EB forming medium has Nuwacell as the basic componentTMhiPSC/hESC medium-ncTarget, and ROCK inhibitor Y27632 and bFGF are additionally added; the concentration of the mother liquor of the ROCK inhibitor Y27632 is 10 mM; the volume ratio of the mother liquor of the ROCK inhibitor Y27632 to the ncTarget is 1: 1000; the final concentration of the bFGF is 2-6 ng/mL, and 4ng/mL is preferred.
As a specific embodiment, the basic component of the nerve induction medium is DMEM/F12, and additionally NEAA (Non Essential Amino Acid, 100X) accounting for 1% of the total volume, GlutaMAX (100X) accounting for 1% of the total volume (available from GIBCO/Invitrogen Cat. No. 35050061), N2 (100X) (N-2 additive) accounting for 1% of the total volume, heparin (preferably 1. mu.g/mL) accounting for 0.5-2. mu.g/mL, and penillin-streptomycin (100X) accounting for 1% of the total volume are added.
In a specific embodiment, the basic components of the neural differentiation medium are DMEM/F12 and Neurobasal medium, the volume ratio of the DMEM/F12 and Neurobasal medium is 1:1, and the base components further comprise B27 with vitamin na (50 ×), N2(100 ×), NEAA (100 ×), GlutaMAX) (1%), penicilin-streptomycin (100 ×), 2-5 μ g/ml-mercaptoethanol (100 ×), preferably 3.5 μ g/ml, and 1-4 μ g/ml insulin solution (preferably 2.5 μ g/ml), wherein the base components account for 1% of the total volume.
In a specific embodiment, the basic components of the maturation induction medium are DMEM/F12 and Neurobasal medium in a volume ratio of 1:1, and 1% by volume of B27(50 ×), 0.5% by volume of N2(100 ×), 0.5% by volume of NEAA (100 ×), 1% by volume of GlutaMAX (100 ×), 1% by volume of penillin-streptomycin (100 ×), 2-5 μ g/ml of beta-mercaptoethanol, preferably 3.5 μ g/ml, 1-4 μ g/ml of insulin solution, preferably 2.5 μ g/ml, are added.
The innovation of the above technical scheme is that (1) there is no organoid in adherent culture before, and the scheme of starting culture from monolayer adherent cells is not obvious: monolayer cells can uniformly contact with differentiation factors, so that a heterogeneous self-organized structure is difficult to generate due to factor concentration gradient, intercellular interaction and the like, therefore, the brain organoid culture starts from EB (Epstein Barr), and the cells are accumulated on a three-dimensional layer by limiting the shape of the monolayer cultured cells and are supported by extracellular matrix, so that the brain organoid is developed, and the generation thought of the brain organoid is widened; (2) the method comprises the following steps: the whole process of culture of the brain organoid can be completed without transferring the culture position, and the culture steps are simplified;
specifically, a cell specific adhesion area with a specific pattern is formed at the bottom of a cell culture vessel through a patterning technology, pluripotent stem cells can be inoculated on a patterned substrate, after the cells adhere to the wall, a perforated thin film is removed to obtain a cell aggregate with a specific edge shape, the stem cells are induced to be differentiated to a brain organoid on the patterned substrate, and finally a brain organoid model with the thickness of 500-1500 microns and the specific edge shape is formed.
According to another exemplary embodiment of the present invention, a brain organoid model obtained by the method is provided. As a specific implementation of the embodiment of the present invention, the obtained brain organoids are in the shape of a segment of a sphere, i.e., a shape of a sphere cut by a plane, which is a phenotype different from the brain organoids; the periphery is randomly distributed with nerval ring structures, and vacuolated structures can appear around part of brain organoids occasionally.
According to another exemplary embodiment of the present invention, there is provided a use of the brain organoid model in neurotoxicity analysis.
The preparation method of the brain organoid model comprises the steps of obtaining a perforated film, preparing a patterned chip, inoculating pluripotent stem cells on a patterned substrate, differentiating towards a neuroectoderm direction, and finally forming a segment-shaped brain organoid which is adhered to the bottom of a culture dish and has a certain thickness and a specific edge shape, wherein the cell types comprise stem cell-derived neural stem cells, neurons, astrocytes and microglia; the brain organoids can be used for studying brain development, diseases, drug screening, drug neurotoxicity evaluation and the like. Compared with the traditional brain organoid culture method, the invention provides a high-throughput, high-homogeneity and in-situ culture brain organoid model, and provides a novel research tool for related brain organoid research.
The following will describe a brain organoid model of the present application, its preparation method and application in detail with reference to the following embodiments and drawings.
Example 1 culture method of brain organoid model
Preparation method of boundary-limited pluripotent stem cells
Preparing an SU-8 template by using a soft lithography technology, wherein the template is a high-flux microcolumn array, the diameter of each microcolumn is 500 micrometers, the height of each microcolumn is 50 micrometers, and the space between the microcolumns is 1000 micrometers;
mixing a PDMS prepolymer and a curing agent according to a ratio of 10:1, pouring the mixture onto an SU-8 template, vacuumizing to remove bubbles, covering a layer of 0.2mm PMMA plate on the mixture, clamping the PMMA plate by two cover glass sheets and fixing the PMMA plate by a bench vice, placing the mixture in an oven at 80 ℃ for polymerization for 120min, taking out the mixture to recover to room temperature, peeling the perforated PDMS film from the SU-8 template, taking out the film with a diameter of 10mm by using a puncher with a suitable size, adding 1mL of 70% ethanol into a 48-hole plate, placing the film at the bottom of the plate, sucking and removing excess ethanol, and drying the film in the oven at 80 ℃ to obtain the patterned chip shown in FIG. 2 a;
sterilizing the patterned chip under an ultraviolet lamp for 60min, adding 500 μ L of 1 XDPBS to each hole to rinse the chip, removing DPBS, adding 0.2mg/mL of Matrigel (DMEM/F12), and incubating at 37 deg.C for 1 h;
digesting the hiPSCs with 70-80% of fusion degree into single fine particles by using AccutaseCells were resuspended with nctarget, 2% Matrigel DMEM/F12 was aspirated and 1.5X 10 cells were added per well5After the cells adhere to the surface, the PDMS film is carefully removed by forceps, and the pluripotent stem cells with boundary limitation are obtained.
The boundary of the pluripotent stem cell is limited to be circular in shape;
the growth area of the pluripotent stem cells (i.e., the area of each of the perforations of the film) was 0.2mm2；
The spacing between the growth areas of the pluripotent stem cells (i.e., the spacing between two adjacent perforations of the film) is 1.5 mm;
the growth region arrangement mode of the pluripotent stem cells is square vertex arrangement;
the culture system of the brain organoid is a 48-pore plate.
Second, culture method of patterned brain organoid
Replacing EB forming culture medium to culture for 6 days, and the effect after the culture is shown as figure 2 b;
after the cells are thickened, the cells are cultured for 5 days by using a nerve induction culture medium;
absorbing and discarding the original culture medium, carefully rinsing twice with 1 XDPBS, coating the patterned cells with pre-cooled Matrigel, solidifying for 37min in a carbon dioxide incubator at 37 ℃, and adding a neural differentiation culture medium for culturing for 3 days;
replacing the induced maturation culture medium for continuous culture, and observing the formed patterned brain organoids after a period of time;
the development of patterned brain organoids induced by the above method was followed brightly and the results are shown in fig. 3.
Example 2
In this embodiment, the boundary of the pluripotent stem cells is limited to a circular shape; the growth area of the pluripotent stem cells is 0.008 mm2(ii) a The interval between the growth areas of the pluripotent stem cells is 1 mm; the arrangement mode of the growth areas of the pluripotent stem cells is square vertex arrangement; the culture system of the brain organoid is a 48-hole plate; the rest of the procedure was the same as in example 1.
Example 3
In this embodiment, a pluralityThe boundary of the stem-competent cells is limited to a circular shape; the growth area of the pluripotent stem cells is 0.03 mm2(ii) a The interval between the growth areas of the pluripotent stem cells is 1.2 mm; the growth region arrangement mode of the pluripotent stem cells is square vertex arrangement; the culture system of the brain organoid is a 48-pore plate; the rest of the procedure was the same as in example 1.
Example 4
In this embodiment, the boundary of the pluripotent stem cells is limited to a circular shape; the growth area of the pluripotent stem cells is 0.8 mm2(ii) a The interval between the growth areas of the pluripotent stem cells is 2 mm; the arrangement mode of the growth areas of the pluripotent stem cells is square vertex arrangement; the culture system of the brain organoid is a 48-hole plate; the rest of the procedure was the same as in example 1.
Example 5
In this embodiment, the boundary of the pluripotent stem cells is limited to a square shape; the growth area of the pluripotent stem cells is 1mm2(ii) a The interval between the growth areas of the pluripotent stem cells is 2 mm; the arrangement mode of the growth areas of the pluripotent stem cells is square vertex arrangement; the culture system of the brain organoid is a 48-hole plate; the rest of the procedure was the same as in example 1.
Example 6
In this embodiment, the boundary of the pluripotent stem cells is shaped as a regular triangle; the growth area of the pluripotent stem cells is 0.43mm2(ii) a The interval between the growth areas of the pluripotent stem cells is 2 mm; the growth region arrangement mode of the pluripotent stem cells is square vertex arrangement; the culture system of the brain organoid is a 48-hole plate; the rest of the procedure was the same as in example 1.
Example 7
In this embodiment, the boundary of the pluripotent stem cells is limited to a cross shape; the growth area of the pluripotent stem cells is 0.2mm2(ii) a The interval between the growth areas of the pluripotent stem cells is 1 mm; the arrangement mode of the growth areas of the pluripotent stem cells is square vertex arrangement; the culture system of the brain organoid is a 48-hole plate; the rest of the procedure was the same as in example 1.
Example 8
The fruitIn the examples, the boundary of the pluripotent stem cells is limited to a circular shape; the growth area of the pluripotent stem cells is 0.2mm2(ii) a The interval between the growth areas of the pluripotent stem cells is 1 mm; the culture system of the brain organoid is a 24-pore plate; otherwise, the same procedure as in example 1 was repeated.
Example 9
In this embodiment, the boundary of the pluripotent stem cells is limited to a circular shape; the growth area of the pluripotent stem cells is 0.2mm2(ii) a The interval between growth areas of the pluripotent stem cells is 1.5 mm; the perforation arrangement mode is regular hexagon arrangement; the culture system of the brain organoid is a 24-pore plate; the rest of the procedure was the same as in example 1.
Example 10
In this example, the cell adhesion material of the patterned brain organoid chip was Vitronectin, and the rest was the same as in example 1.
Comparative example 1
The boundary limiting area of the pluripotent stem cells in this comparative example was 0.002mm2Otherwise, the same procedure as in example 1 was repeated.
Comparative example 2
The boundary limiting area of the pluripotent stem cells in this comparative example was 5mm2Otherwise, the same procedure as in example 1 was repeated.
Comparative example 3
The spacing between the growth zones of the pluripotent stem cells in this comparative example was 0.1mm, and the rest was the same as in example 1.
Comparative example 4
The spacing between the growth zones of the pluripotent stem cells in this comparative example was 0.05mm, and the rest was the same as in example 1.
Comparative example 5
The cell density at which the brain organoids began to differentiate in this comparative example was 5X 103/cm2Otherwise, the same procedure as in example 1 was repeated.
Comparative example 6
The cell density at which the brain organoids began to differentiate in this comparative example was 5X 107/cm2Otherwise, the same procedure as in example 1 was repeated.
Comparative example 7
The cell adhesion substance of the pluripotent stem cells in this comparative example was 10mg/mL Matrigel, and the rest was the same as in example 1.
Comparative example 8
The cell adhesion material of the pluripotent stem cells in this comparative example was 5mg/mL Matrigel, and the rest was the same as in example 1.
Comparative example 9
The cell adhesion material of the pluripotent stem cells in this comparative example was 0.01mg/mL Matrigel, and the rest was the same as in example 1.
Experimental example 1
Statistics were made on the culture effects of the patterned brain organoids of the above examples 1 to 10 and comparative examples 1 to 9, as shown in table 1, wherein the standard deviation coefficient of variation of the area was calculated by: coefficient of variation C · V ═ (standard deviation SD/Mean) × 100%;
TABLE 1
From the data in table 1, it can be seen that:
in comparative example 1, the boundary limiting area of pluripotent stem cells was 0.002mm2Is smaller than 0.005-3.5 mm in the embodiment of the invention2The range of (2) has the defects of untight adhesion of organoids and easy loss in the process of liquid exchange;
in comparative example 2, the boundary limiting area of pluripotent stem cells was 5mm2Is larger than 0.005-3.5 mm in the embodiment of the invention2The range of (1), has the defect that the patterned brain organoid is difficult to culture and form;
in the comparative example 3, the interval between the growth areas of the pluripotent stem cells is 0.1mm, which is smaller than the range of 0.5-245 mm in the embodiment of the invention, fusion can be caused in the growth increase process of the organoid, and the defect that independent organoids are difficult to form exists;
in the comparative example 4, the interval between the growth areas of the pluripotent stem cells is 0.05mm, which is smaller than the range of 0.5-245 mm in the embodiment of the invention, and the defects that fusion is caused in the growth and growth process of organoids and independent organoids are difficult to form exist;
in comparative example 5, the cell density at which the brain organoids started to differentiate was 5X 103/cm2Is smaller than that in embodiment 10 of the present invention5～ 106Has the disadvantage of too low initial cell density to form organoids of multiple cell types;
in comparative example 6, the cell density at which the brain organoids started to differentiate was 5X 107In/cm, greater than that of inventive example 105～106The range of (a) has the disadvantage that the initial cell density is too high, affecting the subsequent differentiation state;
in comparative example 7, the cell adhesion substance of pluripotent stem cells was 10mg/mL Matrigel, which is larger than the range of 0.1 to 1mg/mL in the examples of the present invention, and there was a disadvantage of inducing cells to differentiate in advance in other directions;
in comparative example 8, the cell adhesion substance of pluripotent stem cells was 5mg/mL Matrigel, which is larger than the range of 0.1 to 1mg/mL in the examples of the present invention, and there was a disadvantage of inducing cells to differentiate in advance in other directions;
in comparative example 9, the cell adhesion substance of the pluripotent stem cells is 0.01mg/mL Matrigel, which is smaller than the range of 0.1-1 mg/mL in the embodiment of the present invention, and the defects that the concentration of the extracellular adhesion substance is too low and the cells are difficult to attach exist;
in the embodiments 1-10 of the present invention, the brain organoid model with thickness of 500-.
Comparing the area and variation coefficient of the patterned brain organoids cultured up to day 25 in example 1 with those cultured by conventional methods; as can be seen from fig. 2c and 2d, the size of the patterned brain organoids cultured to the later stage is substantially the same as that of the conventionally cultured brain organoids, but the uniformity of the brain organoids of the embodiment of the present invention is better than that of the conventionally cultured brain organoids.
In summary, the invention utilizes the boundary limitation of the pluripotent stem cells cultured in a single layer to generate the novel adherent culture globular brain organoid model, the model has the brain-related cell types such as neurons, neural stem cells, astrocytes and the like, can realize the high-flux and one-step construction of the brain organoids with uniform shapes, avoids the risks of fusion and pollution caused by methods and operations in the organoid culture process, can observe the whole growth and development process of the organoids in situ, and is used for the application research of the brain organoids.
Experimental example 2 detection of human brain organoid markers
1. Detection of different cell type markers in brain organoids using immunofluorescence: patterned brain organoids were fixed and sectioned at 25 days in culture. NEXTIN and SOX2 co-staining confirmed neural stem cell differentiation, TUJ and MAP2 identified neuronal differentiation, PAX6 identified the expression of forebrain-related markers in patterned brain organoids.
2. Example 1 construction and characterization of an a β 42 oligomer neurotoxicity evaluation system on patterned substrates: on day 25, Control, 0. mu.M, 1. mu.M, and 5. mu.M of A.beta.42 oligomers (A.beta.42O) QPCR was performed 72 hours after treatment of brain organoids to detect expression of neuron-associated markers. The results are shown in fig. 4, which shows that the human brain organoid is successfully obtained and an evaluation system for the neurotoxicity of the abeta 42 oligomer is constructed in the embodiment of the invention.
Finally, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A method of preparing a brain organoid model, the method comprising:
obtaining a unilamellar adherently growing pluripotent stem cell with boundary limitations;
and adding EB (Epstein-Barr) to the pluripotent stem cells with boundary limitation and monolayer adherent growth to form a culture medium for maintaining and culturing for 4-6 days, culturing for 2-6 days by adopting a nerve induction culture medium, then removing the nerve induction culture medium, adding pre-cooled Matrigel for coating, curing, sequentially replacing a nerve differentiation culture medium for culturing for 3-5 days, and culturing in an induction maturation culture medium for 10-30 days to obtain the brain organoid model.
2. The method of claim 1, wherein the method of obtaining boundary confinement in the boundary-confined unilamellar adherently growing pluripotent stem cells comprises one of molecular imprinting, photolithography, perforated thin film, and differential adhesion.
3. The method for preparing a brain organoid model according to claim 1, wherein the obtaining of unilamellar adherently growing pluripotent stem cells with boundary limitations specifically comprises:
obtaining a film having a plurality of perforations;
placing the film at the bottom of a cell culture plate, and adding a cell adhesion material into the cell culture plate for coating;
and inoculating multifunctional stem cells into the coated cell culture plate, and removing the film after the cells adhere to the wall to obtain the monolayer adherent growth multifunctional stem cells with boundary limitation.
4. The method of claim 3, wherein the dimensions of the thin film are matched to the dimensions of the cell culture plate; the hole shape of each perforation of the film comprises one of a circle, an ellipse, a semicircle, a sector, a triangle, a quadrangle, a pentagon, a hexagon, and an arbitrary polygon; the arrangement of the perforations in the film comprises: square vertex arrangement, regular hexagon arrangement, linear arrangement and random arrangement.
5. The method of claim 3, wherein the cell-adhesion material comprises at least one of Matrigel and Vitronectin; the concentration of the cell adhesion material is 0.1-1 mg/mL.
6. The method of claim 1, wherein the pluripotent stem cells comprise one of human embryonic stem cells or human induced pluripotent stem cells.
7. The method for preparing a brain organoid model according to claim 1, wherein the growing area of the unilamellar anchorage-grown pluripotent stem cells with boundary limitation is 0.005-3.5 mm2And the distance between two adjacent unilamellar adherent growth pluripotent stem cell areas with boundary limitation is 0.5-245 mm.
8. A brain organoid model obtained by the method of any one of claims 1 to 7.
9. The brain organoid model of claim 8, wherein the brain organoid shape in the brain organoid model is a segment of a sphere, i.e., a shape of a sphere cut by a plane; the periphery is randomly distributed with nerval ring structures.
10. Use of the brain organoid model of claim 9 in neuro-mechanical studies, neurological disease models, neuro-drug development, and neurotoxicity analysis.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210185202.2A CN114657127B (en) | 2022-02-28 | 2022-02-28 | Brain organoid model and preparation method and application thereof |
| PCT/CN2023/078353 WO2023160688A1 (en) | 2022-02-28 | 2023-02-27 | Brain organoid model, method for preparing same and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023160688A1 (en) * | 2022-02-28 | 2023-08-31 | 武汉大学 | Brain organoid model, method for preparing same and use thereof |
| CN116731859A (en) * | 2023-05-12 | 2023-09-12 | 武汉大学 | Annular brain organoid model and construction method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981096A (en) * | 2014-05-27 | 2014-08-13 | 东南大学 | Two-layer cell culture system organ chip and preparation method thereof |
| CN112680348A (en) * | 2020-12-31 | 2021-04-20 | 北京大橡科技有限公司 | Organ model construction method based on organ chip and organ model |
| US20210284962A1 (en) * | 2018-06-21 | 2021-09-16 | The Regents Of The University Of California | Generation of a population of hindbrain cells and hindbrain-like organoids from pluripotent stem cells |
| CN113773959A (en) * | 2021-08-20 | 2021-12-10 | 武汉大学 | Organoid culture chip and organoid culture method |
| CN114058587A (en) * | 2021-11-18 | 2022-02-18 | 南京医科大学 | Basal forebrain cholinergic organoids prepared by hPSCs induced differentiation and preparation method thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136185B (en) * | 2017-06-28 | 2021-09-21 | 中国科学院生物物理研究所 | Preparation method and application of brain-like organ device |
| CN108456659B (en) * | 2018-03-14 | 2020-05-05 | 浙江霍德生物工程有限公司 | Preparation method of 3D brain organoid |
| KR102228400B1 (en) * | 2019-09-24 | 2021-03-16 | 고려대학교 산학협력단 | Method for preparing brain organoid |
| CN113136367B (en) * | 2020-01-19 | 2022-09-13 | 中国科学院生物物理研究所 | Preparation method and application of striatum organoid |
| CN112481212A (en) * | 2020-11-27 | 2021-03-12 | 杭州联众医疗科技股份有限公司 | Method for generating brain organoid by using pluripotent stem cells |
| CN114657127B (en) * | 2022-02-28 | 2024-09-13 | 合肥燃音生物科技有限公司 | Brain organoid model and preparation method and application thereof |
-
2022
- 2022-02-28 CN CN202210185202.2A patent/CN114657127B/en active Active
-
2023
- 2023-02-27 WO PCT/CN2023/078353 patent/WO2023160688A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981096A (en) * | 2014-05-27 | 2014-08-13 | 东南大学 | Two-layer cell culture system organ chip and preparation method thereof |
| US20210284962A1 (en) * | 2018-06-21 | 2021-09-16 | The Regents Of The University Of California | Generation of a population of hindbrain cells and hindbrain-like organoids from pluripotent stem cells |
| CN112680348A (en) * | 2020-12-31 | 2021-04-20 | 北京大橡科技有限公司 | Organ model construction method based on organ chip and organ model |
| CN113773959A (en) * | 2021-08-20 | 2021-12-10 | 武汉大学 | Organoid culture chip and organoid culture method |
| CN114058587A (en) * | 2021-11-18 | 2022-02-18 | 南京医科大学 | Basal forebrain cholinergic organoids prepared by hPSCs induced differentiation and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| YUJUAN ZHU等: "In situ generation of human brain organoids on a micropillar array", LAB CHIP ., vol. 17, no. 17, pages 2941 - 2950, XP055629153, DOI: 10.1039/C7LC00682A * |
| 杜鑫程等: "运动相关创伤性脑损伤的新兴三维体外模型 研究进展", 生物医学工程学杂志, vol. 38, no. 4, pages 797 - 804 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023160688A1 (en) * | 2022-02-28 | 2023-08-31 | 武汉大学 | Brain organoid model, method for preparing same and use thereof |
| CN116731859A (en) * | 2023-05-12 | 2023-09-12 | 武汉大学 | Annular brain organoid model and construction method and application thereof |
| CN116731859B (en) * | 2023-05-12 | 2024-04-30 | 武汉大学 | Annular brain organoid model and construction method and application thereof |
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| WO2023160688A1 (en) | 2023-08-31 |
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