Bionic micro-fluidic chip model for co-culture of blood brain barrier and organoid as well as construction method and application of bionic micro-fluidic chip model
By constructing a blood-brain barrier and organoid co-culture model in a microfluidic chip, the problems of mismatched channel shapes and cell interactions in existing technologies have been solved, realizing a highly realistic blood-brain barrier model and expanding its application scope.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-17
- Publication Date
- 2026-04-03
AI Technical Summary
Existing 3D cell-organ-on-a-chip models, when simulating the blood-brain barrier, have channel shapes that do not match capillaries, and cannot construct models of the interaction between the blood-brain barrier and other cells, making it difficult to achieve relevant pathological or normal models.
A biomimetic model of co-culture of blood-brain barrier and organoids was constructed using a microfluidic chip. Circular vascular channels and organoid flow channels were set in the chip, and a needle-like template was used to construct circular vascular channels in the blood-brain barrier flow channels. The blood-brain barrier and organoid models were then cultured in the same chip.
It significantly improves the simulation level of the blood-brain barrier model, expands its application fields, and enables drug sensitivity testing, drug screening, pathological model simulation, biomaterial evaluation, immune co-culture, and tumor invasion or migration studies.
Smart Images
Figure CN121780321A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomimetic microfluidic chip model for co-culturing blood-brain barrier and organoids, its construction method, and its application. Background Technology
[0002] The blood-brain barrier is a unique structure found only in brain capillaries. Compared to ordinary capillaries, they contain not only endothelial cells and pericytes, but also astrocytes. Their terminal feet form a complex network around the endothelial cells and basement membrane, connecting endothelial cells, microglia, and neurons, thus providing a barrier between the brain and the bloodstream. Brain capillaries also possess various intercellular junction proteins and efflux transport proteins, resulting in tight cell connections. Furthermore, brain capillaries lack the fenestration structure found in ordinary capillaries. These multiple factors make it difficult for drugs to cross the blood-brain barrier and enter the brain.
[0003] To study the interactions between different drugs and the blood-brain barrier, various in vitro blood-brain barrier models have been developed, including 2D cell models, animal models, and 3D cell-organ-on-a-chip models. Among these, the 3D cell-organ-on-a-chip model is widely recognized as the most valuable in vitro blood-brain barrier model. The 3D cell culture method can effectively simulate the interaction between cells and the matrix microenvironment, maintaining the natural morphology and function of the cells. Blood-brain barrier models constructed using human-derived cells more closely resemble the characteristics of the human blood-brain barrier and show no interspecies differences compared to animal models. The organ-on-a-chip approach allows for the addition of more cell types to the model and provides dynamic environmental support, further enhancing the model's simulation capabilities.
[0004] Currently, common 3D cell-organ-on-a-chip models used to simulate the blood-brain barrier include multi-layer organ-on-a-chips (MLSBs) and parallel organ-on-a-chips. MLSBs, represented by those from Emulate, have two channels, one vertically and one horizontally, separated by a membrane. However, due to limitations in observation methods and manufacturing processes, these chips typically only have two channels. MLSBs with more channels are difficult to manufacture and inconvenient for microscopic observation. Furthermore, using membranes as the barrier structure deviates significantly from the actual blood-brain barrier structure, raising questions about the accuracy of the simulation. Parallel organ-on-a-chips, on the other hand, avoid these drawbacks. Since the channels and chambers are all on the same plane, the manufacturing difficulty is independent of the number of channels, and they allow for convenient optical and fluorescence observation using a microscope.
[0005] However, parallel organ-on-a-chip still faces several challenges: 1. Due to limitations in chip manufacturing techniques, the endothelial cell flow channels in existing organ-on-a-chip models are typically rectangular, which does not match the actual circular shape of capillaries. 2. The interaction between the blood-brain barrier and other cells is not considered, making it impossible to construct relevant pathological or normal models. Therefore, new 3D cell-organ-on-a-chip models are needed to address these issues. Summary of the Invention
[0006] To address the current limitations of 3D cell-organ-on-a-chip models, this invention provides a biomimetic microfluidic chip model for co-culturing the blood-brain barrier and organoids, along with its construction method and applications. This model features circular channels identical to capillaries for culturing vascular endothelial cells. Both the blood-brain barrier model and organoid models can be constructed simultaneously on the same chip, enabling co-culture and expanding the application areas of the blood-brain barrier model.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first objective of this invention is to provide a method for constructing a biomimetic microfluidic chip model for co-culturing the blood-brain barrier and organoids, which is performed using a microfluidic chip and includes the following steps: A microfluidic chip is provided; the microfluidic chip includes a chip body and a base plate for sealing the lower surface of the chip body. The base plate and the chip body are bonded by bonding, thermoforming, and double-sided adhesive bonding. The lower surface of the chip body is provided with a first culture medium channel, an organoid channel, a blood-brain barrier channel, and a second culture medium channel from one side to the other. The first culture medium channel is used to provide culture medium to the organoid channel, and the second culture medium channel is used to provide culture medium to the blood-brain barrier channel. Each channel is recessed into the interior of the chip body. The upper surface of the chip body is provided with an inlet and an outlet for each channel. The lower surface of the chip body is provided with a liquid reservoir at both ends of the blood-brain barrier channel. The sidewall of the chip body is provided with a channel hole extending into the interior of the chip body. The channel hole communicates with the liquid reservoir and the blood-brain barrier channel, facilitating the preparation of circular vascular channels. The microfluidic chip is pretreated for sterilization. A needle-shaped template is inserted into the channel orifice and extends into the blood-brain barrier channel. Hydrogel is injected into the blood-brain barrier channel. After the hydrogel solidifies, the needle-shaped template is extracted, forming a circular vascular channel with the outer contour of the needle-shaped template within the hydrogel. Brain vascular endothelial cells are injected into the circular vascular channel and uniformly adhered to the inner surface of the circular vascular channel. After culture, a blood-brain barrier model is formed. A reservoir is used to store the culture medium for blood-brain barrier growth. Specifically, the needle-shaped template can be a fine needle. A matrix suspension containing organoids is injected into the organoid channel, causing the matrix suspension to solidify. Organoid culture medium is then injected through the first culture medium channel. After cultivation, an organoid model is formed, resulting in a biomimetic microfluidic chip model co-cultured with the blood-brain barrier and organoids. Preferably, during cultivation, a shaker continuously provides nutrients and shear force stimulation to both the blood-brain barrier model and the organoid model. Once the model matures, experiments can be conducted according to experimental requirements, such as those investigating factors affecting the integrity of the blood-brain barrier, the interaction between the blood-brain barrier and organoids, and the efficacy of drugs on tumor organoids in the presence of the blood-brain barrier.
[0008] As a preferred technical solution, the first culture medium channel, organoid channel, blood-brain barrier channel, and second culture medium channel are parallel to each other, and the width of each channel is substantially the same. Each pair of adjacent channels in the first culture medium channel, organoid channel, blood-brain barrier channel, and second culture medium channel is separated by a microwall. The microwall acts as a limiting structure, ensuring that the hydrogel remains only in the injection channel and does not overflow into other channels, ultimately forming a hydrogel barrier. Further, the hydrogel can be a matrix gel, collagen gel, GelMA hydrogel, or other synthetic hydrogels. Further, the height of the microwall is lower than the depth of each channel, so that the culture medium located in the first and second culture medium channels can penetrate into adjacent channels, providing nutrients for model growth.
[0009] As a preferred technical solution, the depth of the blood-brain barrier channel is greater than the depth of the organoid channel, that is, the blood-brain barrier channel has a larger volume, reserving enough space to facilitate the construction of circular vascular channels.
[0010] As a preferred technical solution, the depths of the first culture medium channel, the organoid channel, and the second culture medium channel are 0.1-1 mm, preferably 0.6 mm; the depth of the blood-brain barrier channel is 0.2-2 mm, preferably 1.2 mm; the diameter of the channel pore is 0.1-1 mm, preferably 0.6 mm; the height of the microwall is 0.05-0.5 mm, preferably 0.3 mm. The thickness of the base plate is 0.1-2 mm, preferably 0.6 mm.
[0011] The second objective of this invention is to provide a biomimetic microfluidic chip model for co-culturing blood-brain barrier and organoids, which is obtained using the construction method described in the first objective above.
[0012] A third objective of this invention is to provide the application of the biomimetic microfluidic chip model of blood-brain barrier and organoid co-culture as described in the second objective in drug sensitivity testing, drug screening, pathological model simulation, biomaterial evaluation, immune co-culture, and tumor invasion or migration.
[0013] The present invention has the following beneficial effects: The biomimetic microfluidic chip model for co-culturing the blood-brain barrier and organoids provided by this invention is based on a microfluidic chip. This microfluidic chip contains organoid channels, blood-brain barrier channels, and corresponding culture medium channels. The sidewall of the chip body has channels communicating with the blood-brain barrier channels. During model building, a needle-like template is used in conjunction with the channel structure to construct circular vascular channels with the outer contour of the needle-like template within the blood-brain barrier channels, thereby forming the blood-brain barrier model within these circular vascular channels. The blood-brain barrier model constructed by this invention has a circular structure similar to capillaries, significantly improving the simulation accuracy compared to existing technologies that use membranes as the barrier structure. Furthermore, this invention, through the co-culturing of the blood-brain barrier model and organoid models, solves the problem of single-cell type co-culturing in existing technologies, significantly expanding the application field of the blood-brain barrier model. Attached Figure Description
[0014] Figure 1 This is a schematic diagram of the structure of a microfluidic chip; Figure 2 This is a bottom view of the chip body; Figure 3 for Figure 2 A cross-sectional view of the chip body (containing needle-shaped template) along the AA direction; Reference numerals: 101-Chip body, 102-Base plate, 1011-First culture medium channel, 1012-Organoid channel, 1013-Blood-brain barrier channel, 1014-Second culture medium channel, 1015-Reservoir, 1016-Channel pore, 1017-Microwall, 1018-Needle-shaped template. Detailed Implementation
[0015] The present invention will be further described below with reference to embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention. Unless otherwise specified, the preparation processes in the following embodiments are conventional methods in the prior art, and therefore will not be described in detail. It should be understood that the terms "upper," "lower," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. The terms "first" and "second" do not represent a specific quantity or order, but are only used for distinguishing names.
[0016] I. Microfluidic Chip Structure like Figure 1The diagram shows a chip body 101 and a substrate 102. The substrate material needs to be biocompatible and have good light transmittance, and can be one of glass, polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), and transparent resin.
[0017] The chip body 101 has flow channels and a liquid reservoir, such as Figure 2 and Figure 3 As shown, the channels are a first culture medium channel 1011, an organoid channel 1012, a blood-brain barrier channel 1013, and a second culture medium channel 1014, separated by microwalls 1017. Both the first and second culture medium channels 1011 and 1014 are culture medium channels, respectively supplying culture medium to the organoid channel 1012 and the blood-brain barrier channel 1013. Channel openings 1016 are provided on the wall surface to form circular vascular channels. Large reservoirs 1015 are provided at the front and rear to store culture medium; after the circular vascular channels are formed, the culture medium in the reservoirs provides nutrients to the blood-brain barrier model located within the circular vascular channels through the channel openings.
[0018] The chip's main material needs to be biocompatible; therefore, the material can be one of the following: polydimethylsiloxane (PDMS), polypropylene (PP), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), or a biocompatible transparent resin. Manufacturing methods include integral injection molding, machining, and 3D printing.
[0019] II. Construction Method of Biomimetic Microfluidic Chip Model for Blood-Brain Barrier and Organoid Co-culture Based on Microfluidic Chips First, prepare cerebral vascular endothelial cells, a collagen gel suspension containing astrocytes, microglia, and pericytes, and a matrix gel suspension containing organoids. Then, begin model construction on a microfluidic chip. First, insert a fine needle with a diameter smaller than the channel aperture 1016 into the blood-brain barrier channel 1013. Then, slowly inject the collagen gel suspension into the inlet pool of the blood-brain barrier channel 1013 until it completely fills the channel. Afterward, place the chip in an incubator to solidify the collagen gel suspension, then remove the needle. This creates a circular vascular channel 1018 within the blood-brain barrier channel 1013.
[0020] Brain vascular endothelial cells were introduced into the circular vascular channels 1018 of the blood-brain barrier channel 1013, and then the chip was flipped to allow the vascular endothelial cells to be evenly distributed on the surface of the circular vascular channels. After static culture for one day, fresh culture medium was slowly introduced into the circular vascular channels 1018, and then a matrix gel suspension was introduced into the organoid channel 1012. The chip was then placed in an incubator and allowed to solidify. The culture medium required for organoids was injected into the first culture medium channel 1011, and the culture medium required for the blood-brain barrier was injected into the second culture medium channel 1014. Finally, an appropriate amount of the culture medium required for the blood-brain barrier was added to the large reservoir 1015.
[0021] The organ-on-a-chip was placed on a shaker, and the shaker continuously provided nutrients and shear force stimulation to the blood-brain barrier model and organoid model by swinging it from side to side. Once the model matured, relevant experiments could be carried out.
[0022] Application Example 1 Drug selectivity testing using a microarray model co-cultured with the blood-brain barrier and organoids. (a) Experimental instruments and consumables The organ-on-a-chip, pipette, constant temperature carbon dioxide incubator, microscope, centrifuge tube, clean bench, refrigerator, etc. described in this application.
[0023] (II) Experimental Reagents Culture medium, digestion solution, anhydrous ethanol, PBS buffer, purified water, matrix gel, collagen suspension.
[0024] (III) Experimental Procedure 1. Microfluidic chip pretreatment Immerse the microfluidic chip in 75% ethanol and use a pipette to inject 75% ethanol into the microchannels, ensuring the alcohol flows through the internal channels. After two minutes, remove the organ-on-a-chip. Then immerse it in PBS buffer and perform the same treatment. Finally, remove the microfluidic chip, place it in a sterile environment, and air dry. Before use, irradiate it under UV light for 30 minutes to complete the chip pretreatment.
[0025] 2. Formation of the blood-brain barrier Prepare a collagen gel suspension containing cerebral vascular endothelial cells and astrocytes, microglia, and pericytes. The density of astrocytes should be 1 × 10⁻⁶. 5 The density of microglia was 1 × 10⁶ cells / mL. 5 Pericytes / mL, pericyte density 5×10⁻⁶ 5 The number of organs per mL was then removed, and a clean, sterile fine needle was inserted through the through-hole. Collagen gel suspension was then injected, and the suspension was observed for any overflow. If no abnormalities were found, the suspension was placed in a 37°C incubator for 1 hour to allow the collagen gel suspension to fully solidify.
[0026] After solidification, remove the fine needle and inject cells at a density of 5 × 10⁻⁶ into the formed circular channel. 5 Brain vascular endothelial cells bEND.3 per mL were first placed in a 37°C incubator for 30 minutes, then the chip was flipped and left to stand for another 30 minutes to ensure that the bEND.3 cells could adhere evenly to the inner surface of the circular channels. Finally, culture medium was added to the channels, and the cells were placed on a shaker for dynamic culture for several days to form a complete blood-brain barrier.
[0027] 3. Blood-brain barrier function test Solutions of dopamine and L-dopamine of equal concentration were prepared and added to the first culture medium channel of the organ-on-a-chip. The concentrations of dopamine and L-dopamine in the blood-brain barrier channel were then measured. The results showed that the concentration of L-dopamine was higher than that of dopamine, consistent with the actual results of selective passage of dopamine and L-dopamine through the human blood-brain barrier. This indicates that the blood-brain barrier model constructed in this application has the same function as the human blood-brain barrier and exhibits selective permeability to relevant drugs.
[0028] Application Example 2 Blood-brain barrier toxicity testing was conducted using a microarray model co-cultured with the blood-brain barrier and organoids. The blood-brain barrier model was constructed using the same steps as in Example 1. First, the transmembrane resistance of the blood-brain barrier model was measured using a transmembrane resistance meter. Under normal conditions, the transmembrane resistance of the blood-brain barrier model was greater than 1000 Ω·cm. 2 Subsequently, methamphetamine was introduced into the first culture medium channel. After a period of time, the transmembrane resistance of the blood-brain barrier model decreased, indicating increased permeability and impaired barrier function, consistent with the test results of methamphetamine in the human blood-brain barrier.
[0029] Application Example 3 Drug susceptibility testing using organ-on-a-chip co-culture of blood-brain barrier and organoids. A blood-brain barrier model was constructed using the same steps as in Example 1. A matrix gel suspension containing glioma organoids was then prepared and introduced into the organoid channels. The system was placed in a 37°C incubator. After the matrix gel solidified, glioma organoid culture medium was introduced into the first culture medium channel. The medium was changed daily until the organoids reached a suitable size, at which point drug sensitivity testing began. Simultaneously, a control group consisting only of glioma organoids was prepared and subjected to the same culture method and drug sensitivity testing.
[0030] Temozolomide and crizotinib solutions were prepared. Temozolomide is an anticancer drug that can cross the blood-brain barrier, while crizotinib cannot. Temozolomide and crizotinib solutions were introduced into organ-on-a-chip systems with blood-brain barrier and organoid models, respectively. Simultaneously, temozolomide and crizotinib solutions were added to well plates containing only organoid models. After a period of time, microscopic observation revealed that glioma organoids in the organ-on-a-chip system infused with temozolomide solution underwent significant apoptosis, while those infused with crizotinib solution showed no significant changes. In the control group of well plates, significant apoptosis was observed in all glioma organoids. This indicates that the blood-brain barrier model was effective, preventing drugs that cannot cross the blood-brain barrier from entering the brain and exerting their effects.
[0031] Application Example 4 NK cell killing assay using organ-on-a-chip co-culture of blood-brain barrier and organoids. Using the same method as in Application Example 3, organ-on-a-chip models of the blood-brain barrier and gliomas were constructed, while a control group without a blood-brain barrier model was prepared. Large numbers of NK cells were introduced into both the organ-on-a-chip and the control group. After a period of time, extensive apoptosis of glioma organoids was observed in both experimental groups, indicating that NK cells can penetrate the blood-brain barrier, migrate into tumor tissue, and kill tumor cells. This is consistent with the killing effect of NK cells on brain tumor cells; therefore, the organ-on-a-chip model of this application can be used for the evaluation of NK cell-based immunotherapies.
[0032] The above description is only a preferred embodiment of this application. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of this application, and these improvements and additions should also be considered within the scope of protection of this application.
Claims
1. A method for constructing a biomimetic microfluidic chip model for co-culturing blood-brain barrier and organoids, characterized in that, It is performed using a microfluidic chip and includes the following steps: A microfluidic chip is provided; the microfluidic chip includes a chip body and a base plate for sealing the lower surface of the chip body; the lower surface of the chip body is provided with a first culture medium channel, an organoid channel, a blood-brain barrier channel and a second culture medium channel sequentially from one side to the other; the upper surface of the chip body is provided with inlets and outlets for each channel; the lower surface of the chip body is provided with reservoirs at both ends of the blood-brain barrier channel; the sidewall of the chip body is provided with a channel hole extending into the interior of the chip body; the channel hole communicates with the reservoir and the blood-brain barrier channel; The microfluidic chip is sterilized before being pretreated. A needle-shaped template is inserted into the channel hole and extends into the blood-brain barrier channel. Hydrogel is injected into the blood-brain barrier channel. After the hydrogel solidifies, the needle-shaped template is extracted, forming a circular vascular channel with the outer contour of the needle-shaped template inside the hydrogel. Brain vascular endothelial cells are injected into the circular vascular channel and allowed to adhere evenly to the inner surface of the circular vascular channel. After culture, a blood-brain barrier model is formed. An organoid matrix suspension containing organoids is injected into the organoid channel to solidify the matrix suspension. The organoid culture medium is then injected through the first culture medium channel. After cultivation, an organoid model is formed, resulting in a biomimetic microfluidic chip model of co-culture of the blood-brain barrier and organoids.
2. The construction method according to claim 1, characterized in that, The first culture medium channel, organoid channel, blood-brain barrier channel, and second culture medium channel are parallel to each other.
3. The construction method according to claim 1, characterized in that, Each pair of adjacent channels in the first culture medium channel, organoid channel, blood-brain barrier channel, and second culture medium channel is separated by a microwall; the height of the microwall is less than the depth of each channel.
4. The construction method according to claim 3, characterized in that, The depth of the blood-brain barrier channel is greater than the depth of the organoid channel.
5. The construction method according to claim 4, characterized in that, The depth of the first culture medium channel, the organoid channel, and the second culture medium channel is 0.1-1 mm; the depth of the blood-brain barrier channel is 0.2-2 mm; the diameter of the channel pore is 0.1-1 mm; and the height of the microwall is 0.05-0.5 mm.
6. The construction method according to any one of claims 1 to 5, characterized in that, The thickness of the base plate is 0.1-2 mm.
7. The construction method according to claim 1, characterized in that, The hydrogel is a matrix gel, collagen gel, or GelMA hydrogel; the collagen gel suspension contains astrocytes, microglia, and pericytes.
8. The construction method according to claim 1, characterized in that, During the culture process, the blood-brain barrier model and organoid model are continuously provided with nutrients and shear force stimulation through a shaker.
9. A biomimetic microfluidic chip model for co-culturing blood-brain barrier and organoids, characterized in that: It is obtained by the construction method as described in any one of claims 1 to 8.
10. The application of the biomimetic microfluidic chip model of blood-brain barrier and organoid co-culture as described in claim 9 in drug sensitivity testing, drug screening, pathological model simulation, biomaterial evaluation, immune co-culture, tumor invasion or migration.