Nerve-blood brain barrier co-culture model and construction method and application thereof

Abstract

The invention relates to the technical field of biological tissue engineering, and discloses a nerve-blood brain barrier co-culture model, which comprises a culture chip, and nerves and blood brain barriers respectively constructed in upper culture micropores and lower culture micropores of the culture chip. Wherein a semi-permeable membrane is arranged between the lower-layer culture micropores and the upper-layer culture micropores of the culture chip; the nerves are obtained by inoculating neurons / brain organs obtained by stem cell differentiation into the upper culture micropores for co-culture, and the blood brain barrier is obtained by inoculating brain microvascular endothelial cells obtained by stem cell differentiation into one side of the semipermeable membrane in the lower culture micropores for co-culture. The co-culture model is higher in biomimetic property, and can better simulate the structure and function of the blood brain barrier, for example, the co-culture model has a barrier function closer to the physiological level, the selective permeability of a small molecule drug is matched with the in-vivo permeation condition, and the co-culture model has the function of a small molecule related transporter. The invention further discloses a construction method and application.

Description

Technical Field

[0001] The present application relates to the field of biological tissue engineering technology, for example, to a neuron / brain organoid-blood-brain barrier co-culture model and its construction method and application. Background Art

[0002] The blood-brain barrier (BBB) ​​is a special neurovascular unit that maintains brain homeostasis. It strictly controls the exchange of substances between the blood and the brain and is an essential functional gatekeeper of the central nervous system.

[0003] The blood-brain barrier regulates the penetration and transport of substances from the blood to the brain and from the brain to the blood, and is necessary for the nutrient delivery of the central nervous system and its communication with peripheral tissues. The blood-brain barrier can also prevent neurotoxic components and pathogens in the plasma from entering the brain, maintain the relative stability of the brain's internal environment, and protect the function of the central nervous system. However, due to the existence of the blood-brain barrier, the penetration efficiency of central nervous system drugs into the brain parenchyma is quite limited, resulting in poor drug treatment effects for brain diseases, while accumulation in other organs and tissues leads to aggravated side effects. The delivery of drugs to the central nervous system across the blood-brain barrier remains a major challenge in the development of neurotherapeutics. In order to study the development of the blood-brain barrier, predict the neurotoxicity of drugs, and predict the effectiveness of central nervous system drugs, the development of a blood-brain barrier model is very necessary.

[0004] Traditional models are mainly animal models, and animal models face the following challenges in drug screening: species differences cause differences in physiological structure or gene expression; screening costs are high and throughput is low; in vivo imaging accuracy is poor. Therefore, more and more in vitro models have emerged. Common in vitro models mainly focus on monolayer culture of immortalized human microvascular endothelial cell lines or primary human brain microvascular endothelial cells on Transwell. Compared with in vivo models using experimental animals, the advantages of in vitro models are: the cells are derived from humans, reducing physiological differences between species; drug screening throughput is high. However, conventional in vitro models still have great limitations, such as the poor biomimetic nature of the model constructed by the immortalized human microvascular endothelial cell line, the cells cannot differentiate normally or perform the function of the blood-brain barrier; primary brain microvascular endothelial cells are difficult to obtain, difficult to proliferate in vitro, and large differences between batches.

[0005] Based on the above problems, there is an urgent need for in vitro models that can replace in vivo experiments and have strong bionics for drug screening. Summary of the invention

[0006] In order to provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is given below. The summary is not an extensive review, nor is it intended to identify key / critical elements or delineate the scope of protection of these embodiments, but rather serves as a prelude to the detailed description that follows.

[0007] The disclosed embodiments provide a neuron / brain organoid-blood-brain barrier co-culture model and a construction method and application thereof, so as to obtain an in vitro model that can replace in vivo experiments and has strong bionics.

[0008] In some embodiments, the nerve-blood-brain barrier co-culture model includes: a culture chip, including multiple culture wells, and each culture well includes a lower culture micropore and an upper culture micropore that are connected up and down, and a semipermeable membrane is arranged between the lower culture micropore and the upper culture micropore; the nerve is obtained by inoculating neurons and / or brain organoids obtained by stem cell differentiation into the upper culture micropore for co-culture; the blood-brain barrier is obtained by inoculating brain microvascular endothelial cells obtained by stem cell differentiation into the lower culture micropore for co-culture.

[0009] In some embodiments, the method for constructing the neural organoid-blood-brain barrier co-culture model comprises: differentiating stem cells into neurons or brain organoids, and differentiating stem cells into brain microvascular endothelial cells; inoculating the brain microvascular endothelial cells on the semipermeable membrane side of the lower culture micropores, adding a first culture medium, and culturing; after culturing for 2 to 5 days, removing the first culture medium, inoculating neurons and / or brain organoids into the upper culture micropores, adding a second culture medium to the upper layer, and replacing the lower layer with a fresh first culture medium for co-culturing; wherein the second culture medium is a neuron maturation culture medium or a brain organoid maturation culture medium.

[0010] In some embodiments, the aforementioned nerve-blood-brain barrier co-culture model or the nerve-blood-brain barrier co-culture model constructed by the construction method is used in adeno-associated virus drug screening.

[0011] The present disclosure provides a neural-blood-brain barrier co-culture model and a construction method and application thereof, which can achieve the following technical effects: The neural-blood-brain barrier co-culture model of the disclosed embodiment is a neural and blood-brain barrier co-culture system obtained by using stem cells to differentiate into nerves (e.g., neurons and / or brain organoids) and brain microvascular endothelial cells (iBMECs), and then using neurons (or brain organoids) to co-culture with iBMECs. Compared with conventional in vitro models, the neural-blood-brain barrier co-culture model of the disclosed embodiment is more biomimetic and can better simulate the structure and function of the blood-brain barrier (BBB), such as having a barrier function closer to the physiological level, the selective permeability of small molecule drugs is consistent with the permeation situation in the body, and has small molecule-related transporter functions. In addition, the stem cells of the disclosed embodiment can carry the genetic information of the donor and are used to develop patient-specific disease models or rare disease models, conduct mechanism research and drug screening.

[0012] In addition to the applications in adeno-associated virus drug screening and drug neurotoxicity screening, the application of the disclosed embodiments also has more research application scenarios, which can be used to explore how nerves affect the formation, function and maintenance of the blood-brain barrier, the mechanism of action of brain microvascular endothelial cells in the communication between the central nervous system and peripheral tissues, and how brain microvascular endothelial cells affect neurogenesis and other basic research. In addition, the co-culture model of the disclosed embodiments is an important tool in drug development, providing researchers with drug transport mechanisms in the early stages of drug development, thereby developing targeted delivery drugs; it can be used to evaluate the brain permeability of each candidate drug in the drug screening stage. In short, the co-culture model of the disclosed embodiments provides a good solution for basic research on the blood-brain barrier, detecting the neurotoxicity of drugs, and developing and screening drugs for brain diseases.

[0013] The above general description and the following description are exemplary and explanatory only and are not intended to limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] One or more embodiments are exemplarily described by corresponding drawings, which do not limit the embodiments. Elements with the same reference numerals in the drawings are shown as similar elements, and the drawings do not constitute a scale limitation, and wherein: Figure 1 It is a schematic diagram of the structure of an iPSC-derived neuron-blood-brain barrier co-culture model provided in an embodiment of the present disclosure; Figure 2 It is a schematic diagram of the structure of an iPSC-derived brain organoid-blood-brain barrier co-culture model provided in an embodiment of the present disclosure; Figure 3 is a cross-sectional view of a culture unit of a culture chip provided by an embodiment of the present disclosure; Figure 4 It is a flowchart of a method for constructing an iPSC-derived neural-blood-brain barrier co-culture model provided in an embodiment of the present disclosure; Figure 5a-Figure 5e is a result diagram of the relative gene expression of each marker in the neural progenitor cell differentiation culture after iPSC differentiation in Example 1; Figure 6 is an immunofluorescence staining image of each marker in the neural progenitor cell differentiation culture obtained by iPSC differentiation in Example 1; Figures 7a to 7c is a result diagram of the relative expression of genes of various markers in the midbrain neuron differentiation culture after the neural progenitor cells differentiated in Example 1; Figures 8a to 8d This is a result diagram of the relative expression of genes of markers of brain microvascular endothelial cells differentiated from iPSCs in Example 1; Fig. 9This is an immunofluorescence staining image of the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture in Example 1; Fig.10a and Fig.10b This is a diagram showing the barrier density test results of the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture in Example 1; Fig.11 This is a graph showing the results of the selective permeability test of small molecule drugs in the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture in Example 1; Fig.12 This is a graph showing the results of the P-glycoprotein efflux function test of the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture in Example 1; Fig.13 This is a graph showing the results of AAV titer detection through the blood-brain barrier after AVV drug screening in the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture in Example 1; Fig.14 This is a picture taken by confocal microscope in Example 1 showing the expression of green fluorescent protein in neurons after AVV drug screening in the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture; Fig.15 This is a diagram showing the immunofluorescence staining results of brain organoid slices in the iPSC-derived brain organoid-blood-brain barrier co-culture model constructed in Example 2; Fig.16a and Fig.16b This is a barrier density detection diagram of the iPSC-derived brain organoid-blood-brain barrier co-culture model obtained by co-culture in Example 2; Fig.17 This is a graph showing the results of the selective permeability test of small molecule drugs in the iPSC-derived brain organoid-blood-brain barrier co-culture model obtained by co-culture in Example 2; Fig.18 This is a graph showing the results of the P-glycoprotein efflux function test of the iPSC-derived brain organoid-blood-brain barrier co-culture model obtained by co-culture in Example 2; Fig.19 This is a diagram showing the barrier density test results of the iPSC-derived brain organoid-blood-brain barrier co-culture model obtained by co-culture in Example 2 before and after AVV drug screening; Fig. 20 This is a picture of the green fluorescent protein expression of brain organoids after AVV drug screening in the iPSC-derived brain organoid-blood-brain barrier co-culture model taken with a confocal microscope in Example 2; Fig.21 This is a result diagram of the green fluorescent protein expression of brain organoids after AVV drug screening in the iPSC-derived brain organoid-blood-brain barrier co-culture model detected by flow cytometry in Example 2; Fig. 22 This is a diagram showing the results of immunofluorescence staining of brain organoid sections after AVV drug screening in the iPSC-derived brain organoid-blood-brain barrier co-culture model obtained by co-culture in Example 2.

[0015] Reference numerals: 10. Culture unit; 11. Upper culture micropores; 12. Lower culture micropores; 13. Semipermeable membrane; 14. Upper liquid reservoirs; 15. Lower liquid reservoirs; 16. Horizontal channels; 20. Nerves; 21. Neurons; 22. Brain organoids; 30. Blood-brain barrier. DETAILED DESCRIPTION

[0016] In order to be able to understand the features and technical contents of the embodiments of the present disclosure in more detail, the implementation of the embodiments of the present disclosure is described in detail below in conjunction with the accompanying drawings. The attached drawings are for reference only and are not used to limit the embodiments of the present disclosure. In the following technical description, for the convenience of explanation, a full understanding of the disclosed embodiments is provided through multiple details. However, one or more embodiments can still be implemented without these details. In other cases, to simplify the drawings, well-known structures and devices can be simplified for display.

[0017] The terms "first", "second", etc. in the specification and claims of the embodiments of the present disclosure and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the terms used in this way can be interchanged where appropriate to facilitate understanding of the embodiments of the present disclosure described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions.

[0018] In the embodiments of the present disclosure, the terms "upper", "lower", "inner", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. These terms are mainly intended to better describe the embodiments of the present disclosure and their embodiments, and are not intended to limit the indicated devices, elements, or components to have specific orientations, or to be constructed and operated in specific orientations. Moreover, in addition to being used to indicate orientations or positional relationships, some of the above terms may also be used to indicate other meanings. For example, the term "upper" may also be used to indicate a certain dependency or connection relationship in certain circumstances. For those of ordinary skill in the art, the specific meanings of these terms in the embodiments of the present disclosure can be understood according to specific circumstances.

[0019] Unless otherwise stated, the term "plurality" means two or more.

[0020] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present disclosure may be combined with each other.

[0021] Combination Figures 1 to 3 As shown, a neural-blood-brain barrier co-culture model provided by an embodiment of the present disclosure includes a culture chip and an iPSC-derived neural 20 in an upper culture micropore 11 and a blood-brain barrier 30 in a lower culture micropore 12, respectively constructed in the culture chip. The culture chip 10 includes a plurality of culture wells, each of which includes a lower culture micropore 12 and an upper culture micropore 11 connected up and down, and a semipermeable membrane 13 is provided between the lower culture micropore 12 and the upper culture micropore 11; the iPSC-derived neural 20 is obtained by inoculating neurons and / or brain organs obtained by stem cell differentiation into the upper culture micropore 11 for co-culture, and the blood-brain barrier 30 is obtained by inoculating brain microvascular endothelial cells obtained by stem cell differentiation into one side of the semipermeable membrane 13 in the lower culture micropore 12 for co-culture.

[0022] Stem cells have unlimited proliferation ability and the potential to differentiate into any cell. Therefore, the neural-blood-brain barrier co-culture model of the disclosed embodiment is a neural and blood-brain barrier co-culture system obtained by using stem cells (e.g., human induced pluripotent stem cells iPSC) to differentiate into neurons, brain organoids and brain microvascular endothelial cells (iBMEC), and then using neurons (or brain organoids) to co-culture with iBMEC. Compared with conventional in vitro models, the neural-blood-brain barrier co-culture model of the disclosed embodiment is more biomimetic and can better simulate the structure and function of the blood-brain barrier (BBB), such as having a barrier function closer to the physiological level, and the selective permeability of small molecule drugs is consistent with the permeation situation in the body, and has small molecule-related transporter functions. In addition, the stem cells of the disclosed embodiment can carry the genetic information of the donor, which is used to develop patient-specific disease models or rare disease models, and conduct mechanism research and drug screening.

[0023] The nerve-blood-brain barrier co-culture model of the disclosed embodiment can be used for screening of adeno-associated virus drugs and drug neurotoxicity screening. Moreover, it can also be used to study the interaction mechanism between nerves and the blood-brain barrier, and explore the interaction mechanism of how nerves affect the formation, function and maintenance of the blood-brain barrier. Further, it can also be used to study the mechanism of action of brain microvascular endothelial cells in the communication between the central nervous system and peripheral tissues, and to study the relationship between brain microvascular endothelial cells and neurogenesis (for example, basic research on how brain microvascular endothelial cells affect neurogenesis).

[0024] Optionally, the stem cells include human induced pluripotent stem cells (iPSCs). The nerve-blood-brain barrier co-culture model in this embodiment is defined as an iPSC-derived nerve-blood-brain barrier co-culture model.

[0025] In the disclosed embodiment, in the nerve-blood-brain barrier co-culture model, the nerve 20 includes a neuron 21 or a brain organoid 22, that is, the nerve-blood-brain barrier co-culture model includes a neuron-blood-brain barrier co-culture model (such as Figure 1 ) and brain organoid-blood-brain barrier co-culture models (as shown Figure 2 as shown).

[0026] The transmembrane resistance of the nerve-blood-brain barrier co-culture model of the disclosed embodiment can reach 300 Ω•cm 2 Or above, moreover, the co-culture model has a barrier function that is closer to the physiological level, the selective permeability of small molecule drugs is consistent with the in vivo permeation situation, and it has small molecule-related transporter function.

[0027] Optionally, the horizontal projection of the upper culture micropore 11 is located within the horizontal projection of the lower culture micropore 12. The exposed area of ​​the lower side of the semipermeable membrane is larger than the exposed area of ​​the upper side thereof, so that the integrity and continuity of the blood-brain barrier in the lower culture micropore 12 co-cultured with the neurons and / or brain organoid cells in the upper culture micropore 11 can be ensured, and the performance of the constructed co-culture model can be ensured. Here, the horizontal projection refers to the projection on the horizontal plane.

[0028] Optionally, the horizontal projection of the upper culture microwell 11 is located within the horizontal projection of the lower culture microwell 12 and the horizontal projection of the upper culture microwell 11 is smaller than the horizontal projection of the lower culture microwell 12 .

[0029] The specific structure of the culture chip in the embodiments of the present disclosure is not limited, as long as it can be constructed in conjunction with the construction method to obtain a corresponding nerve-blood-brain barrier co-culture model.

[0030] It is understandable that, in addition to the upper culture micropores and the lower culture micropores, the culture wells also have a liquid storage structure for providing culture fluid to the culture micropores. The liquid storage structure is not limited as long as it can provide culture fluid to the upper culture micropores and the lower culture micropores respectively.

[0031] In some embodiments, Figure 3 As shown, the culture chip includes multiple culture units 10, each culture unit 10 includes an upper culture micropore 11, a lower culture micropore 12, a semipermeable membrane 13, an upper liquid storage hole 14 and a lower liquid storage hole 15, wherein the upper liquid storage hole 14, the upper culture micropore 11 and the lower culture micropore 12 are arranged in sequence in a vertical direction, and a semipermeable membrane 13 is arranged between the upper culture micropore 11 and the lower culture micropore 12, and the horizontal projection of the upper culture micropore 11 is located within the horizontal projection of the lower culture micropore 12; the lower liquid storage hole 15 extends downward from the surface of the culture chip and is connected with the lower culture micropore 12 through a horizontal channel 16.

[0032] The culture chip of this embodiment can operate the lower culture micropores 12 on the front side of the culture chip, reducing the difficulty of inoculating cells in the lower culture micropores 12. At the same time, the horizontal projection of the upper culture micropores 11 is controlled to be located within the horizontal projection of the lower culture micropores 12, so that the exposed area of ​​the lower side of the semipermeable membrane is larger than the exposed area of ​​the upper side, which can ensure the integrity and continuity of the blood-brain barrier in the lower culture micropores 12 co-cultured with the neurons and / or brain organoid cells in the upper culture micropores 11, and ensure the performance of the constructed co-culture model. Here, the horizontal projection refers to the projection on the horizontal plane.

[0033] The number and location of the lower layer liquid storage holes 15 are not limited and can be arranged based on the convenience of operating the lower layer culture micro-holes 12 .

[0034] Optionally, the lower liquid storage holes 15 are arranged in pairs and are located on opposite sides of the upper liquid storage hole 14 respectively.

[0035] In the culture chip of this embodiment, a plurality of culture units 10 are arranged in an array, for example, in a square array. The arrangement spacing of the plurality of culture units is set according to subsequent detection requirements.

[0036] Optionally, the top of the horizontal channel 16 is tilted upward from the side of the lower liquid reservoir 15 to the side of the lower culture micropore 12. When the brain microvascular endothelial cells are inoculated into the lower culture micropore 12 through the lower liquid reservoir 15, the brain microvascular endothelial cells can reach the semipermeable membrane 13 in the lower culture micropore 12 by inverting the culture chip.

[0037] Combination Figure 4 As shown, a method for constructing a nerve-blood-brain barrier co-culture model provided in an embodiment of the present disclosure comprises the following steps: S10, Differentiate stem cells into neurons or brain organoids; Differentiate stem cells into brain microvascular endothelial cells; S20, inoculating brain microvascular endothelial cells on the semipermeable membrane side of the lower culture micropores of the culture chip, adding the first culture solution, and culturing; S30. After culturing for 2 to 5 days, remove the first culture medium, inoculate neurons and / or brain organoids into the upper culture microwells of the culture chip, add the second culture medium to the upper layer, and replace the lower layer with fresh first culture medium for co-culture.

[0038] In the construction method of the disclosed embodiment, stem cells (for example, human induced pluripotent stem cells iPSCs or embryonic stem cells ESCs) are differentiated into neurons (or brain organoids) and brain microvascular endothelial cells by utilizing their potential to differentiate into any cells. The brain microvascular endothelial cells are then inoculated on one side of the semipermeable membrane of the lower culture micropores of the culture chip (i.e., the lower surface of the semipermeable membrane) to construct a blood-brain barrier. After culturing for 2 to 5 days (during this period, the brain microvascular endothelial cells begin to form a dense barrier), neurons and / or brain organoids are inoculated into the upper culture micropores of the culture chip (i.e., the upper surface of the semipermeable membrane) for co-culture to construct corresponding neurons, or brain organoids, or mixed cells of neurons and brain organoids. The construction method is simple and easy.

[0039] In some embodiments, the step S10 of differentiating stem cells into neurons or brain organoids includes: S11, differentiating stem cells into neural progenitor cells, and differentiating neural progenitor cells into neurons; or, S12, differentiating stem cells into embryoid bodies, and then subjecting the embryoid bodies to neural induction, and then differentiating to obtain brain organoids, and finally performing maturation culture to obtain brain organoids. In this embodiment, the stem cells include human induced pluripotent stem cells (iPSCs) or embryonic stem cells ESCs.

[0040] In some embodiments, the step S10 of differentiating stem cells into brain microvascular endothelial cells includes: S13, differentiating stem cells into endothelial progenitor cells, and maturing and culturing the endothelial progenitor cells to obtain brain microvascular endothelial cells. In this embodiment, the stem cells include human induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0041] In the disclosed embodiment, steps S11, S12 and S13 have no sequence and can be performed simultaneously.

[0042] In this embodiment, when the stem cells are differentiated, the corresponding differentiation fluid can be selected according to the specific stem cell type and the differentiation target. The differentiation fluid is not limited.

[0043] Optionally, in step S11, the neural progenitor cell differentiation solution used to differentiate the stem cells into neural progenitor cells includes: basal medium IMDM, basal medium F12, MEM non-essential amino acid solution, B-27 additive, N-2 additive, Wnt signaling pathway activator, BMP signaling pathway inhibitor, and TGF-β type 1 receptor inhibitor. Among them, the volume ratio of basal medium IMDM to basal medium F12 is 0.8-1.2:1, the volume ratio of MEM non-essential amino acid solution to basal medium F12 is 0.02:1, and the volume ratio of B-27 additive to basal medium F12 is 0.04:1; the volume ratio of N-2 additive to basal medium F12 is 0.02:1, the concentration of Wnt signaling pathway activator is 3-8 μM, the concentration of BMP signaling pathway inhibitor is 0.1-1 μM, and the concentration of TGF-β type 1 receptor inhibitor is 5-30 μM.

[0044] Specifically, the neural progenitor cell differentiation fluid of this embodiment includes, by volume percentage, 48% IMDM, 48% F12, 1% MEM non-essential amino acid solution, 2% B-27 additive and 1% N-2 additive, as well as 3~8 μM Wnt signaling pathway activator, 0.1~1 μM BMP signaling pathway inhibitor, and 5~30 μM TGF-β type 1 receptor inhibitor.

[0045] Among them, Wnt signaling pathway activators are not limited, for example, CHIR99021, CHIR98014, Activin A, etc.

[0046] BMP signaling pathway inhibitors are not limited, for example, LDN193189 Tetrahydrochloride, Dorsomorphin, etc.

[0047] TGF-β type 1 receptor inhibitors are not limited, for example, SB431542, LY364947, etc. BMP signals have the ability to determine the direction of nerve development, and Wnt signals play an important role in maintaining the proliferation ability of nerve cells. During embryonic development, cell clusters adjacent to nerve-related cell groups will secrete a series of BMP pathway inhibitory proteins, such as Noggin, etc., to promote the differentiation of the cell group in the direction of nerves. After that, the adjacent cell groups will begin to secrete ligand proteins of Wnt pathway receptors, activate and maintain certain Wnt pathway activities, thereby promoting the formation of neural progenitor cells and maintaining their self-proliferation ability. Therefore, by adding BMP pathway inhibitors and Wnt pathway receptor activators to the culture medium, some key stimuli in the development process can be simulated in vitro, thereby inducing the formation of neural progenitor cells and maintaining their long-term stable proliferation.

[0048] Optionally, in step S11, in the process of differentiating neural progenitor cells into neurons, the neuronal differentiation fluid used includes: Neurobasal culture medium, MEM non-essential amino acid solution or / and GlutaMAX additive, small molecules required for neuronal directional differentiation, and neurotrophic factors. Specifically, the neurons in this embodiment are midbrain neurons, and the differentiation fluid includes 96% Neurobasal culture medium, 1% GlutaMax, 2% B-27 additive, 1% N-2 additive, 0.1~1 μM ascorbic acid, 50~300 ng / ml sonic hedgehog factor, and 50~300 ng / ml FGF8B.

[0049] Optionally, in step S12, in the process of differentiating stem cells into brain organoids, the brain organoid differentiation fluid used includes: basal culture medium DMEM / F12 and Neurobasal culture medium, MEM non-essential amino acid solution, GlutaMAX additive, B-27 additive, B-27 additive without vitamin A, N-2 additive, small molecule inducer heparin sodium solution, insulin, etc. In this embodiment, brain organoid differentiation is divided into four stages. The first stage is the embryoid body formation stage, and the culture medium in this stage is mTeSRPLUS culture medium (Stemcell technology, 100-0276). The second stage is the neural induction stage, and the brain organoid differentiation fluid in this stage includes: 97% basal culture medium DMEM / F12, 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, 1~20 ng / ml small molecule inducer heparin sodium. The third stage is the brain organoid differentiation stage. The brain organoid differentiation fluid at this stage includes 47.5% basal medium DMEM / F12 and 47.5% Neurobasal medium, 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, 2% B-27 additive without vitamin A, and small molecule inducer 1~30ng / mL insulin. The fourth stage is the brain organoid maturation culture stage. The culture fluid at this stage includes 47.5% basal medium DMEM / F12 and 47.5% Neurobasal medium, 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, 2% B-27 additive, and small molecule inducer 1~30ng / mL insulin.

[0050] In some embodiments, in step S13, the stem cells are differentiated into endothelial progenitor cells, and the endothelial progenitor cells are matured and cultured to obtain brain microvascular endothelial cells; specifically, it includes: S130, differentiating the stem cells into mesoderm cells, differentiating the mesoderm cells into endothelial progenitor cells, and maturing and culturing the endothelial progenitor cells into brain microvascular endothelial cells to obtain brain microvascular endothelial cells. In this embodiment, the stem cells include human induced pluripotent stem cells (iPSCs).

[0051] Optionally, in step S130, in the process of differentiating stem cells into mesoderm cells, the differentiation fluid used includes: basal culture medium DMEM / F12, culture medium additives MEM non-essential amino acid solution and GlutaMAX additive, Wnt signaling pathway activator. Specifically, the differentiation fluid of this embodiment contains 98% basal culture medium DMEM / F12, 1% MEM non-essential amino acid solution, 1% GlutaMAX additive, and 3~8 μM Wnt signaling pathway activator. Among them, the Wnt signaling pathway activator includes CHIR99021 and CHIR98014.

[0052] Optionally, in differentiating mesoderm cells into endothelial progenitor cells, the differentiation fluid used includes: endothelial cell culture medium, culture medium additive B-27 additive, small molecule inducer retinoic acid, cytokine FGF-2, wherein the volume ratio of endothelial cell culture medium to B-27 additive is 48-50:1, the concentration of retinoic acid is 5-30 μM, and the concentration of FGF-2 is 5-30 ng / mL. Specifically, the differentiation fluid of this embodiment includes 98% (vt.) endothelial cell culture medium (ScienCell, #1001), 2% (vt.) B-27 additive, 5-30 μM retinoic acid, and 5-30 ng / ml FGF-2. Retinoic acid and FGF2 are both important substances secreted during brain development, which can induce brain angiogenesis, vascular-related pericyte differentiation, and the development and maturation of vascular networks, so their addition will promote the formation and maturation of brain-related microvascular endothelium.

[0053] Optionally, in the process of maturation culture from endothelial progenitor cells to brain microvascular endothelial cells, the differentiation solution used includes: endothelial cell culture medium, B-27 additive, small molecule inducer retinoic acid, Y27632, cytokine FGF-2. The differentiation solution of this embodiment includes 98% (vt.) endothelial cell culture medium (ScienCell, #1001), 2% (vt.) B-27 additive, 5~30 μM Y27632, 5~30 μM retinoic acid, and 5~30 ng / ml FGF-2.

[0054] In step S20 of the embodiment of the present disclosure, brain microvascular endothelial cells are inoculated on one side of the semipermeable membrane in the lower culture microwells of the culture chip, and a first culture solution is added and cultured, which specifically includes: S21, adding matrix gel to the lower culture microwells to coat the semipermeable membrane of the culture chip; S22, digesting the brain microvascular endothelial cells, inoculating the cells into the lower culture microwells of the culture chip, and placing the culture chip upside down in an incubator so that the cells fall on the semipermeable membrane, and culturing for 1 to 3 hours; S23, placing the culture chip upright, and adding the first culture solution to both the upper culture microwells and the lower culture microwells; S24, replacing the first culture solution in the upper and lower culture microwells every day.

[0055] Among them, the first culture medium is a brain microvascular endothelial cell culture medium. The brain microvascular endothelial cell culture medium of this embodiment includes: endothelial cell culture medium, B-27 additive, Y27632, retinoic acid and FGF-2, wherein the volume ratio of the endothelial cell culture medium to the B-27 additive is 48-50:1, the concentration of Y27632 is 5-30 μM, the concentration of retinoic acid is 5-30 μM, and the concentration of FGF-2 is 5-30 ng / mL.

[0056] Specifically, the first culture medium includes: 98% (vt.) endothelial cell culture medium (ScienCell, #1001), 2% (vt.) B-27 additive, 5~30 μM Y27632, 5~30 μM retinoic acid, 5~30 ng / mL FGF-2. Among them, the endothelial cell culture medium uses ScienCell's products, which contain more components that promote the formation and function of endothelial cells, such as VEGF vascular endothelial growth factor, bFGF basic fibroblast growth factor, and hydrocortisone that promotes endothelial function barrier function. Therefore, the brain microvascular endothelial cell culture medium based on this basic culture medium can promote the differentiated endothelial cells to have better proliferation ability and cell-to-cell cell junction protein expression level in addition to the effects of retinoic acid and FGF2, two factors related to brain microvascular differentiation, and promote the formation of a more functional and better BMEC barrier.

[0057] In this embodiment, Figure 3 As an example of the culture chip shown in FIG. Figure 3 The lower layer storage hole 15 of the culture chip shown in the figure adds the matrix gel into the lower layer culture microwell 12; similarly, in step S22, Figure 3 The lower liquid reservoir wells 15 of the culture chip shown are used to seed cells into the lower culture microwells 12 of the culture chip.

[0058] In step S30 of the embodiment of the present disclosure, neurons are inoculated into the upper culture microwells, and a second culture medium is added for co-culture, which specifically includes: S31, removing the first culture medium in the upper culture microwells, coating the upper microwells of the culture chip with a neural cell coating medium, digesting the neurons obtained in step S10 (or step S11) and inoculating them into the upper culture microwells, and adding the second culture medium into the upper culture microwells; wherein the second culture medium is a neuron maturation culture medium.

[0059] In step S30 of the embodiment of the present disclosure, brain organoids are inoculated into the upper culture microwells, and a second culture medium is added for co-culture, which specifically includes: S32, removing the first culture medium in the upper culture microwells, transferring the brain organoids obtained in step S10 (or step S12) to the upper culture microwells, and adding the second culture medium to the upper culture microwells; wherein the second culture medium is a brain organoid maturation culture medium.

[0060] Optionally, the neurons in step S31 include but are not limited to midbrain neurons, forebrain neurons, motor neurons, sensory neurons, etc. Wherein, the second culture medium is a neuron maturation culture medium.

[0061] Optionally, if the neurons are midbrain neurons, the second culture medium is a midbrain neuron maturation medium, including 97% Neurobasal, 1% GlutaMax, 2% B-27 supplement, 0.1~1 μM Ascorbic Acid, 5~30 μM DAPT, 0.1~5 mM db-cAMP, 50~300 ng / ml GDNF, and 1~30 ng / ml TGF-β.

[0062] Optionally, the brain organoids of step S32 include but are not limited to forebrain organoids, midbrain organoids, cerebral cortex organoids, non-directed differentiated brain organoids, etc. Among them, the second culture medium is a brain organoid maturation culture medium. The brain organoids of this embodiment are non-directed differentiated brain organoids, and the second culture medium specifically includes 47.5% basal culture medium DMEM / F12 and 47.5% Neurobasal, culture medium additives 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additives, 2% B-27 additives, and 1~30 ng / ml small molecule inducer insulin.

[0063] In some embodiments, the neurons include midbrain neurons, forebrain neurons, motor neurons or sensory neurons, and the brain organoids include midbrain organoids, forebrain organoids, cerebral cortex organoids or non-directed differentiated brain organoids. The co-culture model constructed in this embodiment is a neuron / brain organoid-blood-brain barrier co-culture model, which can be used to develop corresponding physiological or disease models.

[0064] In the method for constructing the nerve-blood-brain barrier co-culture model of the disclosed embodiment, the number of co-culture days can be determined according to the experimental purpose.

[0065] The disclosed embodiments also provide an application of a nerve-blood-brain barrier co-culture model in adeno-associated virus (AAV) drug screening.

[0066] Gene therapy is a new type of treatment, that is, by introducing exogenous normal genes into target cells to correct defects and abnormal genes, so as to achieve the purpose of treating genetic diseases; or inserting exogenous genes into patient receptor cells through transgenic technology, so that the exogenous genes express products, thereby treating diseases. AAV capsid protein is the key to viral recognition and binding to cell surface receptors. There are hundreds of naturally occurring AAV capsid proteins, which we usually call different AAV serotypes. They differ in amino acid sequence and spatial structure and have different tropisms for specific tissues or cells. Like most drugs for nervous system diseases, AAV drugs also face the problem of limited penetration efficiency due to the obstruction of the blood-brain barrier. Since the neural-blood-brain barrier co-culture model (e.g., iPSC-derived neural-blood-brain barrier co-culture model) of the disclosed embodiment has good biomimetic properties and better simulates the structure and function of the blood-brain barrier (BBB), it can be applied to AAV drug screening, and the screening results have good reference significance.

[0067] The co-culture model of the disclosed embodiment is constructed on a culture chip having a plurality of culture wells, so that screening tests of multiple drugs and multiple concentrations can be performed on the co-culture model, which has the advantage of high-throughput screening / research.

[0068] Specific examples are given below to specifically illustrate the neuron / brain organoid-blood-brain barrier co-culture model and its construction method and application in the embodiments of the present disclosure.

[0069] Example 1 iPSC-derived neuron-blood-brain barrier co-culture model A method for constructing an iPSC-derived neuron-blood-brain barrier co-culture model comprises the following steps: S101. Differentiate iPSCs into neural progenitor cells to obtain neural progenitor cell differentiation culture; the neural progenitor cell differentiation medium used includes 47.5% IMDM, 47.5% F12, 1% MEM non-essential amino acid solution, 2% B-27 additive, 1% N-2 additive, 3~8 μM CHIR99021, 0.1~1 μM LDN193189 Tetrahydrochloride, and 5~30 μM SB431542. S102. Differentiate neural progenitor cells into midbrain neuronal cells. When the differentiation reaches 7 to 14 days, obtain midbrain neuronal differentiation culture; wherein the midbrain neuronal differentiation fluid used includes: Neurobasal, 1% GlutaMax, 2% B-27 additive, 1% N-2 additive, 0.1~1 μM Ascorbic Acid, 50~300 ng / ml Sonic Hedgehog, 50~300 ng / ml FGF8b. S103. iPSCs were differentiated into mesoderm and endothelial progenitor cells step by step, and endothelial progenitor cells were matured into brain microvascular endothelial cells to obtain brain microvascular endothelial cells. The differentiation medium used for mesoderm differentiation included 98% DMEM / F12, 1% MEM non-essential amino acid solution and 1% GlutaMAX additive, 3~8 μM CHIR99021; the differentiation medium used for endothelial progenitor cell differentiation included: endothelial cell culture medium (ScienCell, #1001), 2% B-27 additive, 5~30 μM retinoic acid, 5~30 ng / ml FGF-2; the culture medium used for maturation culture included endothelial cell culture medium (ScienCell, #1001), 2% B-27 additive, 5~30 μM Y27632, 5~30 μM retinoic acid, 5~30 ng / ml FGF-2.

[0070] S104. Inoculate brain microvascular endothelial cells on one side of the semipermeable membrane in the lower culture micropores, add the first culture medium, and culture; wherein the first culture medium is a brain microvascular endothelial cell maturation culture medium, specifically including endothelial cell culture medium (ScienCell, #1001), 2% B-27 additive, 5~30 μM Y27632, 5~30 μM retinoic acid, and 5~30 ng / ml FGF-2.

[0071] S105. After 2 to 5 days of culture, the first culture medium is removed, the midbrain neurons are inoculated into the upper culture microwells, the second culture medium is added, and the lower microwells are replaced with fresh first culture medium for co-culture. The second culture medium is a midbrain neuron maturation medium, which specifically includes Neurobasal, 1% GlutaMax, 2% B-27 additive, 0.1~1 μM AscorbicAcid, 5~30 μM DAPT, 0.1~5 mM db-cAMP, 50~300 ng / ml GDNF, and 1~30 ng / ml TGF-β.

[0072] In this Example 1, the schematic diagram of the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture is as follows: Figure 1As shown, the blood-brain barrier composed of iPSC-derived midbrain neurons and brain microvascular endothelial cells is separated by a semipermeable membrane and is located in the upper culture microwells and the lower culture microwells of the culture chip, respectively.

[0073] In this Example 1, the expression of markers in the differentiation products obtained in each step was detected using qPCR technology and immunofluorescence technology. Figure 5a-Figure 5e and Figure 6 As shown, the iPSC marker OCT4 (as shown in Figure 2A) in the neural progenitor cell (NPC) differentiation culture after iPSC differentiation in step S101 Figure 5a ) and NANOG (as Figure 5b ) was downregulated, and the neural progenitor cell marker NESTIN (as Figure 5c as shown), PAX3 (as Figure 5d ) and PAX6 (as Figure 5e In the differentiation culture of neural progenitor cells (NPC) in step S102 and midbrain neurons (MN) differentiated from S105, the expression of neural progenitor cell marker NESTIN was downregulated, and the expression of midbrain neuron markers NR4A2 and TH was upregulated ( Figure 7a , Figure 7b and Figure 7c Steps S103, S104 and S105 are to differentiate iPSCs into brain microvascular endothelial cells (BMECs) that highly express brain microvascular endothelial cell markers such as CDH5, TJP1, ABCG2, and ABCB1 (e.g. Figures 8a to 8d In this Example 1, brain microvascular endothelial cell marker ZO-1 and neuron marker TUJ1 were used to perform immunofluorescence staining on the iPSC-derived midbrain neuron-blood-brain barrier co-culture model. The results are shown in Fig. 9 As shown, tight junctions are formed between brain microvascular endothelial cells in the co-culture model to form a blood-brain barrier; neurons can also differentiate normally.

[0074] In this Example 1, the barrier function of the iPSC-derived midbrain neuron-blood-brain barrier co-culture model obtained by co-culture was tested. Fig.10a and Fig.10b As shown in Figure 2, the co-culture model has good barrier tightness, with a transmembrane electrical resistance (TEER) of approximately 300 Ω•cm 2 , the apparent permeability coefficient P of the small molecule tracer fluorescent yellow app Stably below 5×10 -7 cm / s. Fig.11 As shown in Figure 2, the selective permeability of small molecule drugs in the co-culture model is consistent with the in vivo permeability. The apparent permeability coefficient P of the low permeability drug Atenolol in the co-culture model is appSignificantly lower than the highly osmotic drug Metoprolol.

[0075] In this Example 1, the P-glycoprotein efflux function of the co-culture model was detected. The P-glycoprotein substrate Rhodamine 123 (Rhodamine 123) or cyclosporine A + Rhodamine 123 (CsA + Rhodamine 123) was added to the blood-brain barrier side of the co-culture model, and the apparent permeability coefficient P of Rhodamine 123 was detected after 1 to 5 hours. app , the results are as follows Fig.12 As shown, after the co-addition of cyclosporine A, a competitive inhibitor of rhodamine 123, the permeability of rhodamine 123 increased, that is, the efflux of rhodamine 123 decreased, indicating that the co-culture model has the efflux function of P-glycoprotein.

[0076] In this Example 1, AAV screening was performed using an iPSC-derived midbrain neuron-blood-brain barrier co-culture model that was co-cultured for 7 days. Two serotypes of AAV were used in this Example 1, namely AAV-3 and AAV-9. Both AAVs carry the expression sequence of green fluorescent protein. The infected cells will express green fluorescent protein and produce spontaneous fluorescence. The two serotypes of AAV were diluted separately in the brain microvascular endothelial cell maturation culture medium, and the AAV titers were adjusted to the same titer and added to the lower culture microwells of the culture chip. At the same time, the upper culture microwells of the culture chip were replaced with fresh second culture fluid and culture continued. After the infection was completed, the culture fluid in the upper microwells was collected, and the titers of the two serotypes of AAV in the supernatant were detected using qPCR technology. The results are as follows. Fig.13 As shown, the penetration rate of AAV-9 is significantly higher than that of AAV-3. After collecting the supernatant, the co-culture model is replaced with fresh culture medium and cultured for 1-5 days. The neurons are labeled using immunofluorescence technology, and the expression of green fluorescent protein in the neurons is recorded ( Fig.14 ). It can be seen that AAV-9 has a stronger ability to pass through the blood-brain barrier and infect neurons than AAV-3.

[0077] Example 2 A method for constructing an iPSC-derived brain organoid-blood-brain barrier co-culture model comprises the following steps: S201. Differentiate iPSC into brain organoids, wherein, on days 0 to 2, the culture medium is mTeSR PLUS culture medium (Stemcell technology, 100-0276); on days 3 to 10, the differentiation medium includes basal culture medium DMEM / F12, culture medium additives 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, and small molecule inducer 1-20 ng / ml heparin sodium; on days 10 to 17, the differentiation medium includes basal culture medium DMEM / F12 and Neurobasal, culture medium additives 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, 2% B-27 additive without vitamin A, and small molecule inducer 1-30 ng / ml insulin; after day 18, the culture medium includes basal culture medium DMEM / F12 and Neurobasal, culture medium additives 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, 2% B-27 supplement, small molecule inducer 1~30 ng / ml insulin. Brain organoids that have begun to mature can be used to build co-culture models.

[0078] S202, iPSCs were gradually differentiated into mesoderm and endothelial progenitor cells, and endothelial progenitor cells were matured into brain microvascular endothelial cells to obtain brain microvascular endothelial cells. The differentiation medium used for mesoderm differentiation included 98% DMEM / F12, 1% MEM non-essential amino acid solution and 1% GlutaMAX additive, 3~8 μM CHIR99021; the differentiation medium used for endothelial progenitor cell differentiation included: endothelial cell culture medium (ScienCell, #1001), 2% B-27 additive, 5~30 μM retinoic acid, 5~30 ng / ml FGF-2; the culture medium used for maturation culture included endothelial cell culture medium (ScienCell, #1001), 2% B-27 additive, 5~30 μM Y27632, 5~30 μM retinoic acid, 5~30 ng / ml FGF-2.

[0079] S203. Inoculate brain microvascular endothelial cells on one side of the semipermeable membrane in the lower culture micropores, add the first culture medium, and culture; wherein the first culture medium is a brain microvascular endothelial cell maturation culture medium, specifically including endothelial cell culture medium (ScienCell, #1001), 2% B-27 additive, 5~30 μM Y27632, 5~30 μM retinoic acid, and 5~30 ng / ml FGF-2.

[0080] S204, after culturing for 2 to 5 days, remove the first culture medium, inoculate the mature brain organoid obtained in step S201 into the upper culture microwell, add the second culture medium, and co-culture. The second culture medium is a brain organoid maturation culture medium, which specifically includes basal culture medium DMEM / F12 and Neurobasal, culture medium additives 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, 2% B-27 additive, and small molecule inducer 1~30 ng / ml insulin.

[0081] Schematic diagram of the iPSC-derived brain organoid-blood-brain barrier co-culture model obtained by co-culture in Example 2, as shown in Figure 2 As shown, the barrier formed by brain organoids and iPSC-derived brain microvascular endothelial cells is separated by a semipermeable membrane and is located in the upper and lower micropores of the culture chip, respectively.

[0082] In this Example 2, the section immunofluorescence technique was used to detect the markers of brain organoids in the iPSC-derived brain organoid-blood-brain barrier co-culture model after 10 days of co-culture. Fig.15 As shown, the brain organoids obtained in steps S201 and S204 contain neural progenitor cells (PAX6-positive cells), mature neurons (NeuN-positive cells), and astrocytes (S100B and GFAP double-positive cells). In addition, neural progenitor cells aggregate to form the ventricular zone (VZ), and some neural progenitor cells migrate outward from the ventricular zone and differentiate into neurons to form the subventricular zone (SVZ).

[0083] In this Example 2, the barrier function of the iPSC-derived brain organoid-blood-brain barrier co-culture model after 10 days of co-culture was tested. Fig.16a and Fig.16b As shown in Figure 2, the co-culture model has good barrier tightness, with a transmembrane electrical resistance (TEER) of approximately 300Ω•cm 2 , the apparent permeability coefficient P of the small molecule tracer fluorescent yellow app Stably below 5×10 -7 cm / s. Fig.17 As shown, the selective permeability of small molecule drugs in the co-culture model is consistent with the in vivo permeability. The apparent permeability coefficient of the low permeability drug Atenolol in the co-culture model is significantly lower than that of the high permeability drug Metoprolol.

[0084] In this Example 2, the P-glycoprotein efflux function of the co-culture model was detected. The P-glycoprotein substrate Rhodamine 123 (Rhodamine 123) or cyclosporine A + Rhodamine 123 (CsA + Rhodamine 123) was added to the blood-brain barrier side of the co-culture model, and the apparent permeability coefficient P of Rhodamine 123 was detected after 1 to 5 hours. app , the results are as follows Fig.18 As shown, after the co-addition of cyclosporine A, a competitive inhibitor of rhodamine 123, the permeability of rhodamine 123 increased, that is, the efflux of rhodamine 123 decreased, indicating that the co-culture model has the function of P-glycoprotein efflux. That is, the iPSC-derived brain organoid-blood-brain barrier co-culture model in this Example 2 has the function of P-glycoprotein efflux.

[0085] In this Example 2, AAV screening was performed using an iPSC-derived brain organoid-blood-brain barrier co-culture model that was co-cultured for 7 days. Two serotypes of AAV were used in Example 2, namely AAV-3 and AAV-9. Both AAVs carry the expression sequence of green fluorescent protein. The infected cells will express green fluorescent protein and produce spontaneous fluorescence. The AAV was diluted in the brain microvascular endothelial cell maturation culture medium and added to the lower culture microwells of the culture chip. The upper culture microwells of the culture chip were replaced with fresh brain organoid culture medium. The infection time was about 2-6 days. After the infection was completed, the barrier density was tested, and the expression of green fluorescent protein in the brain organoids was recorded, such as Fig.19 As shown in Figure 2, after AAV infection, the barrier tightness is intact. Fig. 20 and Fig.21 As shown, confocal microscopy was first used to evaluate the expression of green fluorescent protein in brain organoids. The ability of AAV-9 to infect brain organoids after penetrating the blood-brain barrier was significantly stronger than that of AAV-3 ( Fig. 20 Then, flow cytometry was used to detect the proportion of cells expressing green fluorescent protein (EGPF + ), the results showed that AAV-9 is more capable of passing through the blood-brain barrier and infecting brain organoids than AAV-3 ( Fig.21 ), which is consistent with the confocal microscopy results. In addition, AAV-9 infected brain organoids were sliced, neurons were labeled using immunofluorescence, and images were taken using a confocal microscope, such as Fig. 22 As shown, AAV-9 crosses the blood-brain barrier and infects neurons in brain organoids.

[0086] The percentages mentioned in the embodiments of the present disclosure are volume percentages unless otherwise specified.

[0087] The components in each differentiation solution and culture medium in the embodiments of the present disclosure can be purchased.

[0088] In the embodiment of the present disclosure, the upper layer and the lower layer are defined by taking the semi-permeable membrane as a reference.

[0089] In the embodiment of the present disclosure, during the barrier function detection of the co-culture model, a cell transmembrane resistance meter was used to apply a low-frequency, low-amplitude alternating current to the cell barrier and detect the corresponding voltage change to calculate and obtain the resistance level of the cell barrier, thereby providing feedback on the permeability level of the cell barrier to ions and the density of the barrier; the apparent permeability coefficient P of the small molecule tracer fluorescent yellow was used. app By adding fluorescent yellow tracer to one side of the cell barrier and detecting the amount of tracer that penetrates through the barrier and accumulates on the opposite side per unit time, the permeation rate per unit time is calculated, the cell barrier's ability to block small molecules is evaluated, and the density of the cell barrier is also fed back.

[0090] The above description and the accompanying drawings sufficiently illustrate the embodiments of the present disclosure to enable those skilled in the art to practice them. Other embodiments may include structural and other changes. The embodiments represent only possible variations. Unless explicitly required, individual components and functions are optional, and the order of operations may vary. Portions and features of some embodiments may be included in or replace portions and features of other embodiments. The embodiments of the present disclosure are not limited to the structures described above and shown in the accompanying drawings, and various modifications and changes may be made without departing from the scope thereof. The scope of the present disclosure is limited only by the appended claims.

Claims

1. A nerve-blood-brain barrier co-culture model, characterized in that: include: The culture chip comprises a plurality of culture wells, and each culture well comprises a lower culture microwell and an upper culture microwell which are connected up and down, and a semipermeable membrane is arranged between the lower culture microwell and the upper culture microwell; The neural part is obtained by inoculating neurons and / or brain organoids obtained by differentiation of stem cells into the upper culture microwells for co-culture; The blood-brain barrier is obtained by inoculating brain microvascular endothelial cells differentiated from stem cells into the lower culture microwells for co-culture.

2. The nerve-blood-brain barrier co-culture model according to claim 1, characterized in that: Has one or more of the following properties: Transmembrane resistance can reach 300 Ω•cm 2 or above; The apparent permeability coefficient of the small molecule tracer fluorescent yellow is less than 5×10 -7 cm / s; The permeability selectivity of small molecule drugs is consistent with the permeation situation in vivo; It has small molecule-related transporter function.

3. The nerve-blood-brain barrier co-culture model according to claim 1, characterized in that: The horizontal projection of the upper culture microwell is located within the horizontal projection of the lower culture microwell.

4. The method for constructing a nerve-blood-brain barrier co-culture model according to any one of claims 1 to 3, characterized in that: include: Differentiate stem cells into neurons or brain organoids, and differentiate stem cells into brain microvascular endothelial cells; The brain microvascular endothelial cells are seeded on the semipermeable membrane side of the lower culture microwells, and the first culture solution is added for culture; After culturing for 2 to 5 days, the first culture medium is removed, neurons and / or brain organoids are inoculated into the upper culture microwells, the second culture medium is added to the upper layer, and the lower layer is replaced with fresh first culture medium for co-culture; wherein the second culture medium is a neuron maturation medium or a brain organoid maturation medium.

5. The construction method according to claim 4, characterized in that: Differentiating stem cells into neurons or brain organoids, including: differentiating stem cells into neural progenitor cells, differentiating neural progenitor cells into neurons; or differentiating stem cells into embryoid bodies, then subjecting the embryoid bodies to neural induction, then differentiating them to obtain brain organoids, and finally subjecting them to maturation culture to obtain brain organoids; Differentiating stem cells into brain microvascular endothelial cells includes: differentiating stem cells into endothelial progenitor cells, and maturing and culturing the endothelial progenitor cells to obtain brain microvascular endothelial cells.

6. The construction method according to claim 4, characterized in that: The first culture medium includes endothelial cell culture medium, B-27 additive, Y27632, retinoic acid and FGF-2, wherein the volume ratio of endothelial cell culture medium to B-27 additive is 48-50:1, the concentration of Y27632 is 5-30 μM, the concentration of retinoic acid is 5-30 μM, and the concentration of FGF-2 is 5-30 ng / mL.

7. The construction method according to claim 4, characterized in that: The neuron maturation medium includes a midbrain neuron maturation medium; the midbrain neuron maturation medium includes 97% Neurobasal, 1% GlutaMax, 2% B-27 supplement, 0.1~1 μM Ascorbic Acid, 5~30 μM DAPT, 0.1~5 mM db-cAMP, 50~300 ng / ml GDNF, and 1~30 ng / ml TGF-β; Alternatively, the brain organoid maturation medium includes a non-directed differentiation brain organoid maturation medium; the non-directed differentiation brain organoid maturation medium includes 47.5% basal medium DMEM / F12 and 47.5% Neurobasal, culture medium additives 1% MEM non-essential amino acid solution, 1% GlutaMax, 1% N-2 additive, 2% B-27 additive, and 1~30 ng / ml small molecule inducer insulin.

8. The construction method according to claim 5, 6 or 7, characterized in that: In the process of differentiating stem cells into endothelial progenitor cells, the differentiation fluid used includes endothelial cell culture medium, B-27 additive, retinoic acid and FGF-2, wherein the volume ratio of endothelial cell culture medium to B-27 additive is 48-50:1, the concentration of retinoic acid is 5-30 μM, and the concentration of FGF-2 is 5-30 ng / mL; and / or, In the process of maturation culture of endothelial progenitor cells to obtain brain microvascular endothelial cells, the first culture medium is used for maturation culture.

9. The construction method according to claim 5, 6 or 7, characterized in that: Neurons include, but are not limited to, midbrain neurons, forebrain neurons, motor neurons, or sensory neurons; and / or, Brain organoids include, but are not limited to, forebrain organoids, midbrain organoids, cerebral cortex organoids, or non-directed differentiated brain organoids.

10. Use of the stem cell-derived neural-blood-brain barrier co-culture model as described in any one of claims 1 to 3, or the stem cell-derived neural-blood-brain barrier co-culture model obtained by the construction method of the stem cell-derived neural-blood-brain barrier co-culture model as described in any one of claims 4 to 9 in adeno-associated virus drug screening and / or drug neurotoxicity screening.

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