A microfluidic organ chip for modeling dual brain region cell differentiation and methods of use thereof

Abstract

The application relates to the field of organ chips, and particularly discloses a microfluidic organ chip for simulating cell differentiation in double brain regions and a use method thereof. The microfluidic organ chip comprises a plurality of culture modules; a single culture module comprises a first chip layer and a second chip layer; the first chip layer is provided with a culture chamber and a first flow channel; the first flow channel is used for fluid circulation; the culture chamber is communicated with the first flow channel; the first chip layer is further provided with an inoculation hole used for inoculating the culture chamber; the second chip layer is provided with a second flow channel; the liquid outlet end of the second flow channel is in a closed state when the second chip layer is used; the flow rate ratio of the fluid in the first flow channel to the fluid in the second flow channel is (2-20):1; and the culture chamber is communicated with the second chip layer through a communication hole. The organ chip can simulate the development characteristics of the dorsal-ventral cortex of the brain, so that the same cell can be simultaneously differentiated into the ventral and dorsal multiple brain region cortex organoids in the culture chamber.

Description

Technical Field

[0001] This application relates to the field of organ-on-a-chip, and more specifically, to a microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions and a method of using it. Background Technology

[0002] Organ-on-a-chip, short for microfluidic organ-on-a-chip, is a three-dimensional cell culture device that includes a microenvironment composed of cells surrounding a corresponding organ, secretions, and physical forces. Compared with traditional drug screening models, organ-on-a-chip can better simulate the function of real organs and avoids the species differences that exist in animal models.

[0003] Currently, there are significant differences between human and animal brains in terms of volume, cell type, layered distribution, gene expression, and morphology. Brain organoids, brain structures formed by the directed differentiation of human stem cells, can mimic the early embryonic brain development process and structural characteristics in vitro, showing great promise in studying the etiology, potential mechanisms, and drug screening of neuropsychiatric diseases. Although brain organoid technology has made great progress, especially in the establishment of neurological disease models and drug screening, it still has many shortcomings in terms of cellular composition, brain region complexity, metabolic characteristics, and long-term culture. In particular, for mental diseases such as autism and epilepsy, there is often an imbalance in the ratio of excitatory and inhibitory neurons in the cerebral cortex, and there is an urgent need to establish convenient and rapid methods for the differentiation of multi-region brain organoids.

[0004] In recent years, there have been reports of establishing complex brain organoid models by differentiating dorsal and ventral forebrain organoids and then fusing them later, or by knocking in the SHH gene into human embryonic stem cells using genetic engineering techniques and inducing the differentiation of organoids from multiple brain regions. However, these methods are complex, time-consuming, cannot fully simulate the cell interactions during brain development, and require technicians to master complex genetic engineering techniques.

[0005] Therefore, to overcome the challenges of the lack of dual-brain region structures (dorsolateral forebrain and ventral forebrain) and the complexity of differentiation methods in brain organoids, it is crucial to develop a microfluidic brain organoid chip that is easy to operate and can simulate the developmental characteristics of the dorsal-ventral cortex of the brain. Summary of the Invention

[0006] This application provides a microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions and a method for using the same organ-on-a-chip. The organ-on-a-chip can simulate the developmental characteristics of the dorsal-ventral cortex of the brain, enabling the same cell to differentiate into organoids of multiple brain regions, including the ventral and dorsal cortex, in a culture chamber.

[0007] Firstly, the microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions provided in this application adopts the following technical solution:

[0008] A microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions, the organ-on-a-chip comprising multiple culture modules;

[0009] Each of the aforementioned cultivation modules includes a first chip layer and a second chip layer.

[0010] The first chip layer is provided with a culture chamber and a first flow channel. The first flow channel is for fluid circulation, and the flow rate of the fluid in the first flow channel does not exceed 10 μL / min.

[0011] The culture chamber is connected to the first flow channel, and the first chip layer is also provided with an inoculation hole for inoculating into the culture chamber;

[0012] The second chip layer is provided with a second flow channel. When the second chip layer is in use, the liquid outlet of the second flow channel is in a closed state. The flow rate ratio of the fluid in the first flow channel to the fluid in the second flow channel is (2-20):1.

[0013] The culture chamber is connected to the second chip layer through a connecting hole.

[0014] By strictly controlling the fluid flow rates in the first and second channels, and sealing the outlet of the second channel during use, the liquid in the second channel can only flow towards the first channel through the connecting hole. Since the liquid in the first channel itself has a certain flow rate, the fluid in the first channel and the fluid entering from the connecting hole have a faster flow rate near the connecting hole, meaning a faster turnover rate. The fluid in the second chip layer will not stagnate after entering the first chip layer. Therefore, when organoids are seeded near the connecting hole, only those on the side closest to the connecting hole can come into contact with the fluid in the second chip layer, creating a three-dimensional concentration gradient change starting from the connecting hole. This allows the organoids to be regulated by different fluids, resulting in different developmental directions. As a result, the same cell can simultaneously differentiate into ventral and dorsal multi-brain cortical organoids in the culture chamber.

[0015] Furthermore, the flow rate of the fluid in the first flow channel is 1-2.5 μL / min;

[0016] Furthermore, the flow rate of the fluid in the first flow channel is 1.5-2 μL / min.

[0017] Furthermore, the flow rate of the fluid in the first channel can be 1 μL / min, 1.5 μL / min, 1.8 μL / min, 2 μL / min, etc., with the optimal flow rate being 2 μL / min;

[0018] Further research revealed that controlling the fluid velocity in the first channel between 1.5-2 μL / min can better create a concentration gradient with the fluid entering from the connecting hole, allowing organoids to be regulated in different ways and promoting cell differentiation into different organoids.

[0019] Furthermore, the diameter of the connecting hole is 0.08-0.12 mm.

[0020] Furthermore, the diameter of the connecting hole can be 0.08mm, 0.1mm, 0.12mm, etc., with the optimal diameter being 0.1mm.

[0021] The above technical solution further optimizes the aperture of the connecting hole to strictly control the flow rate of fluid in the second channel through the connecting hole into the culture chamber. This creates a favorable concentration gradient with the fluid in the first channel near the connecting hole, ensuring that organoids can be regulated differently and develop in different directions. If the aperture of the connecting hole is too large or too small, a favorable concentration gradient cannot be formed near the connecting hole, thus affecting organoid development.

[0022] Furthermore, the diameter of the inoculation hole is 3.5-4.5 mm.

[0023] Furthermore, the diameter of the inoculation hole can be 3.5mm, 3.8mm, 4mm, 4.5mm, etc., with the optimal diameter being 4mm.

[0024] Furthermore, the first chip layer is also provided with a sealing plug, which is used to seal the inoculation hole.

[0025] Using inoculation wells with a diameter of 3.5-4.5 mm further facilitates inoculation by operators, making it simple and convenient. After inoculation, the inoculation wells are sealed with corresponding sealing plugs to reduce the impact of external factors on organoid differentiation in the culture chamber.

[0026] Furthermore, support columns are symmetrically arranged on both sides of the connecting hole within the culture chamber.

[0027] By adopting the above technical solution, the setting of the support column can not only further regulate the fluid flow rate to a certain extent, but also provide some support for the differentiated organoids, effectively preventing the organoids from moving along the flow direction of the fluid after development and differentiation.

[0028] Furthermore, the materials of the first and second chip layers include one of PDMS, PMMA, PC, and COC.

[0029] Furthermore, the materials for the first and second chip layers are preferably PDMS.

[0030] Furthermore, the first chip layer and the second chip layer are combined using either integral molding or layered bonding.

[0031] Further optimization involves bonding the first chip layer and the second chip layer together after plasma treatment.

[0032] By adopting the above technical solution and selecting appropriate materials, the cost can be effectively reduced and the applicability of organ-on-a-chip can be guaranteed. By selecting appropriate methods to process and combine the first chip layer and the second chip layer, the assembly is simple and practical.

[0033] Secondly, the method for using a microfluidic organ-on-a-chip to simulate cell differentiation in two brain regions provided in this application adopts the following technical solution:

[0034] A method for using a microfluidic organ-on-a-chip to simulate cell differentiation in two brain regions includes the following steps:

[0035] After disinfection, the first and second chip layers are connected to the pipeline.

[0036] Remove healthy EBs after induction and inoculate them into the culture chamber. Add Matrigel, adjust the position of the EBs, and incubate until the Matrigel solidifies.

[0037] Different culture media are introduced into the first chip layer and the second chip layer respectively. After the second chip layer is filled, the liquid outlet of the second chip layer is sealed.

[0038] Adjust the flow rate of the first and second chip layers and continue culturing the organ-on-a-chip for 6-8 days;

[0039] Replace the first and second chip layers, and continue culturing for 6-8 days.

[0040] In summary, this application has the following beneficial effects:

[0041] 1. This application involves the structural design of organ-on-a-chip, strict control of the fluid flow rate in the first and second channels, and sealing the outlet of the second channel during use. Because the outlet of the second channel is sealed, the liquid in the second channel can only flow towards the first channel through the connecting hole. Since the liquid in the first channel itself has a certain flow rate, the fluid in the first channel and the fluid entering from the connecting hole have a faster flow rate near the connecting hole, i.e., a faster turnover rate. The fluid in the second chip layer will not stagnate after entering the first chip layer. Therefore, when organoids are seeded near the connecting hole, only those on the side closest to the connecting hole can come into contact with the fluid in the second chip layer, creating a three-dimensional concentration gradient change starting from the connecting hole. This allows the organoids to be regulated by different fluids, resulting in different developmental directions. Thus, the same cell can simultaneously differentiate into ventral and dorsal multi-brain cortical organoids in the culture chamber.

[0042] 2. The support columns not only further regulate the fluid flow rate, but also provide some support for the differentiated organoids, effectively preventing them from moving along the flow direction of the fluid. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram of the structure of Embodiment 1 of this application;

[0045] Figure 2 This is a schematic diagram of the structure of Embodiment 1 of this application;

[0046] Figure 3 This is a partial structural diagram of Embodiment 1 of this application, mainly to show the connecting hole;

[0047] Figure 4 This is a partial structural diagram of Embodiment 2 of this application, mainly to show the culture chamber and the support column;

[0048] Figure 5 This shows the trend of reaction concentration change when the flow rate ratio is 2:1 in the simulation experiment;

[0049] Figure 6 The trend of reaction concentration change when the flow rate ratio is 20:1 in the simulation experiment;

[0050] Figure 7 The growth of organoids cultured using the organ-on-a-chip method of Example 2;

[0051] Figure 8 This refers to organoids that have been fluorescently labeled.

[0052] Explanation of reference numerals in the attached figures:

[0053] 1. Culture module; 11. First chip layer; 12. Second chip layer; 13. Culture chamber; 14. First flow channel; 15. Inoculation well; 16. Second flow channel; 17. Connecting hole; 18. Adjustment column; 19. Sealing plug; 20. Liquid inlet; 21. Liquid outlet; 22. Liquid inlet hole; 23. Liquid outlet hole. Detailed Implementation

[0054] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0055] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, features referred to as "first" or "second" may explicitly or implicitly include one or more of that feature. Specifically, the materials described in this application are all commercially available materials, specifically: PDMS is polydimethylsiloxane, PMMA is polymethyl methacrylate, PC is polycarbonate, and COC is a cyclic olefin copolymer.

[0056] Example 1

[0057] A microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions is disclosed. The organ-on-a-chip includes multiple culture modules 1. Each culture module 1 includes a first chip layer 11 and a second chip layer 12. The first chip layer 11 and the second chip layer 12 are made of PDMS. The first chip layer 11 is provided with a culture chamber 13 and a first flow channel 14 for fluid flow. The first chip layer 11 and the second chip layer 12 are bonded together after plasma treatment. Plasma treatment is the surface treatment of the first chip layer and the second chip layer using a plasma surface treatment machine, which is common knowledge in the art and will not be described in detail in this application.

[0058] The culture chamber 13 is connected to the first flow channel 14. The culture chamber 13 is cylindrical in shape. The first chip layer 11 has an inlet 20 for fluid to flow in and an outlet 21 for fluid to flow out. Both the inlet 20 and the outlet 21 are connected to the first flow channel 14. The first flow channel 14 is 3mm high and 3mm wide, and the fluid flows from right to left. The top of the first chip layer 11 also has an inoculation hole 15 with a diameter of 4mm. The inoculation hole 15 is connected to the culture chamber 13 to facilitate inoculation into the culture chamber 13. The first chip layer 11 is also provided with a sealing plug 19, which is used to seal the inoculation hole 15 so that the inoculation hole 15 can be sealed after inoculation.

[0059] The second chip layer 12 is provided with a second flow channel 16. The second chip layer 12 has an inlet hole 22 for fluid to flow in and an outlet hole 23 for fluid to flow out. Both the inlet hole 22 and the outlet hole 23 are connected to the second flow channel 16. The second flow channel 16 is 1 mm wide and 0.5 mm high. When the second chip layer 12 is in use, the outlet hole 23 of the second flow channel 16 is closed. The culture chamber 13 is connected to the second chip layer 12 through a connecting hole 17 with a diameter of 0.1 mm.

[0060] Example 2

[0061] The only difference from Example 1 is that support columns are symmetrically arranged on both sides of the connecting hole 17 in the culture chamber 13. A 0.3mm gap is provided between the ends of the two support columns near the liquid outlet 21. A 0.3mm gap is left between the outer wall of the support column and the side wall of the culture chamber 13. The support column is 1.5mm high. The rest of the structure is the same as in Example 1.

[0062] In the actual cell culture process of organ-on-a-chip, since the first and second chip layers are indistinguishable in appearance, to further study and demonstrate the concentration gradient change near the connecting holes when the flow rate ratio of the fluid in the first channel to that in the second channel is (2-20):1, the material transport and concentration diffusion are simulated using the COMSOL Multiphysics microfluidic module. In fluid flow, viscous forces and relative inertial forces generated by shearing on the velocity isosurface dominate, and this phenomenon is very obvious. The Reynolds number (Re) describing the ratio of these two forces is usually very low, so such fluids are usually laminar. In many cases, the Stokes flow model (Re«1) is used for simulation. Diffusion is usually a slow process in microfluidic systems, which affects the transport of chemical substances within the microfluidic system. Microfluidic modules are particularly suitable for studying fluid flow transmission and mass transfer processes at the microscale.

[0063] The COMSOL multiphysics simulation software was used to simulate two physical fields: laminar flow and rarefied material transport. The geometric model was based on a 3D chip model, and the fluid material was defined as water with a viscosity 100 times its normal value and a diffusion coefficient of 0.01 m. 2 / s, the concentration in the second channel is 0.5 mol / m 3 The concentration in the culture chamber flow channel is 0 mol / m³. 3 The locations of the inlet and outlet of the first flow channel and the inlet and outlet of the second flow channel are defined. In the laminar flow physics field, the flow velocities of the first and second flow channels are defined as 0.008 m / s and 0.004 m / s, respectively, i.e., a velocity ratio of 2:1. The inlet and outlet of the first flow channel, the inlet and outlet of the second flow channel, and wall features are also defined. Using the system's custom mesh generation, concentration streamline diagrams and concentration surfaces are plotted, and cross-sectional views are extracted to obtain the reaction concentration variation trend, as shown below. Figure 5 As shown.

[0064] The COMSOL multiphysics simulation software was used to simulate two physical fields: laminar flow and rarefied material transport. The geometric model was based on a 3D chip model, and the fluid material was defined as water with a viscosity 100 times its normal value and a diffusion coefficient of 0.01 m. 2 / s, the concentration in the second channel is 0.5 mol / m 3 The concentration in the culture chamber is 0 mol / m³. 3 The locations of the inlet and outlet of the first flow channel and the inlet and outlet of the second flow channel are defined. In the laminar flow physics field, the flow velocities of the first and second flow channels are defined as 0.08 m / s and 0.004 m / s, respectively, i.e., a velocity ratio of 20:1. The inlet and outlet of the first flow channel, the inlet and outlet of the second flow channel, and wall features are also defined. Using the system's custom mesh generation, concentration streamline diagrams and concentration surfaces are plotted, and cross-sectional views are extracted to obtain the reaction concentration variation trend, as shown below. Figure 5 As shown.

[0065] A method for using a microfluidic organ-on-a-chip to simulate cell differentiation in two brain regions includes the following steps:

[0066] Cell culture:

[0067] 1. Coat the 6-well cell culture dish with Matrigel (8 μg / mL) one hour in advance.

[0068] 2. Transfer 12 mL of mTeSR™1 culture medium into a 15 mL centrifuge tube.

[0069] 3. Remove the iPSC / hESC cells to be revived from liquid nitrogen and place them in a 37°C water bath for 2-3 minutes until the ice crystals are almost completely melted. After sterilizing with 75% alcohol, transfer them to a biosafety cabinet. Take 1 mL of mTeSR™1 medium and add it dropwise to the cryovial. Then transfer all the cells to the centrifuge tube from step 2 and centrifuge at 200g and 20°C for 5 minutes. Discard the supernatant and add 1 mL of mTeSR™1 medium. Gently pipette and resuspend until the cells are visibly fine (avoid over-piping to prevent cell suspension).

[0070] 4. Remove the pre-coated 6-well plate, discard the culture medium, add 2 mL of mTeSR™1 culture medium, and transfer the cells from step 3 into the 6-well plate.

[0071] 5. Gently shake the 6-well plate to mix the cells, then place it in a 37°C, 5% CO2 incubator.

[0072] 6. Change the culture medium daily until the cell density reaches 80%, then you can passage the cells.

[0073] Cell passage:

[0074] 1. Wash away residual culture medium and dead cells with DPBS, repeat twice.

[0075] 2. Add 1 mL of ReLeSR™ to each well and incubate at room temperature for 2-3 minutes until some cells begin to detach when the culture dish is gently tapped.

[0076] 3. Discard ReLeSR™, add 1 mL of mTeSR™1 medium, continue to tap the culture dish until most clones detach, transfer the cells to EP tubes, gently pipette until small particles are visible to the naked eye, and passage at a ratio of 1:30-1:40.

[0077] Formation of the embryoid body:

[0078] 1. Digest cells to the size of visible fine particles according to cell passage method, and transfer them to low-adsorption 6-well plates (no Matrigel coating required).

[0079] 2. After gently shaking, transfer to a 37°C, 5% CO2 incubator and incubate for at least 24 hours without disturbance to ensure embryoid formation (EB).

[0080] 3. Change the mTeSR™1 medium daily for 5 days.

[0081] Embryomorphic body induction of differentiation:

[0082] 1. Change the culture medium and use the STEMdiff™ Cerebral Organoid Kit to induce differentiation.

[0083] 2. Change the culture medium daily and culture for 5 consecutive days until the edges of the EB are smooth and clear.

[0084] EB microarray seeding and amplification (differentiation) culture:

[0085] 1. After sterilizing the first and second chip layers and other devices with 75% alcohol, place them in a biosafety cabinet and connect the tubing.

[0086] 2. Remove the healthy EB from the area near the inoculation well after induction is complete, aspirate excess culture medium, add an appropriate amount of Matrigel, adjust the position of the EB, and incubate in a 37°C, 5% CO2 incubator for 20 minutes to allow the Matrigel to solidify.

[0087] 3. Different culture media were introduced into the first chip layer and the second chip layer respectively. The first chip layer was Neurobasal+Glu+NEAA+N2+B27-A+EGF+FGF+P / S. The second chip layer was supplemented with SHH+Pur. After the second chip layer was filled, its outlet hole was blocked.

[0088] 4. Adjust the flow rate of the microfluidic system. The flow rate of the culture medium in the first channel is 2 μL / min, and the flow rate of the culture medium in the second channel is 0.1 μL / min. Place the organ-on-a-chip in the incubator and continue to culture for 7 days.

[0089] Promote maturity cultivation:

[0090] Replace the culture medium for the first and second chip layers: The first chip layer is Neurobasal + Glu + NEAA + N2 + B27 + BDNF + NT3 + GDNF + P / S. The second chip layer is based on this with the addition of SHH. Keep other conditions unchanged and continue culturing for 7 days.

[0091] Organoid growth status, such as Figure 7 As shown.

[0092] After organoid differentiation and maturation culture on the microarray, the organoids were removed from the microarray and immunofluorescence was used to confirm successful differentiation of organoids in multiple brain regions. PAX6 was used to label the dorsal forebrain, and NKX2.1 was used to label the ventral forebrain. (Specific details are as follows...) Figure 8 As shown.

[0093] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions, characterized in that, The organ-on-a-chip includes multiple culture modules (1); Each of the culture modules (1) includes a first chip layer (11) and a second chip layer (12). The first chip layer (11) is provided with a culture chamber (13) and a first flow channel (14), the first flow channel (14) is for fluid flow, and the flow rate of the fluid in the first flow channel (14) does not exceed 10 μL / min; The culture chamber (13) is connected to the first flow channel (14), and the first chip layer (11) is also provided with an inoculation hole (15) for inoculating into the culture chamber (13); The second chip layer (12) is provided with a second flow channel (16). When the second chip layer (12) is in use, the liquid outlet (23) of the second flow channel (16) is in a closed state. The ratio of the flow velocity of the fluid in the first flow channel (14) to the flow velocity of the fluid in the second flow channel (16) is (2-20):1; The culture chamber (13) is connected to the second chip layer (12) through a connecting hole (17); the diameter of the connecting hole (17) is 0.08-0.12 mm.

2. The microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions according to claim 1, characterized in that: The flow rate of the fluid in the first flow channel (14) is 1-2.5 μL / min.

3. The microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions according to claim 2, characterized in that: The inoculation hole (15) has a diameter of 3.5-4.5 mm.

4. The microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions according to claim 3, characterized in that: The first chip layer (11) is also provided with a sealing plug (19), which is used to seal the inoculation hole (15).

5. The microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions according to claim 1, characterized in that: The culture chamber (13) is symmetrically provided with support columns on both sides of the connecting hole (17).

6. The microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions according to any one of claims 1-5, characterized in that: The materials of the first chip layer (11) and the second chip layer (12) include one of PDMS, PMMA, PC, and COC.

7. The microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions according to claim 6, characterized in that: The first chip layer (11) and the second chip layer (12) are combined using a one-piece molding or layer bonding method.

8. The method of using the microfluidic organ-on-a-chip for simulating cell differentiation in two brain regions as described in any one of claims 1-7, characterized in that: Includes the following steps: After disinfection, the first chip layer (11) and the second chip layer (12) are connected to the pipeline; Take out the healthy EB after the induction is completed and inoculate it into the culture chamber (13), add Matrigel, adjust the position of the EB and incubate until Matrigel solidifies; Different culture media are introduced into the first chip layer (11) and the second chip layer (12) respectively. After the second chip layer (12) is filled, the liquid outlet hole (23) of the second chip layer (12) is sealed. Adjust the flow rate of the first chip layer (11) and the second chip layer (12) and continue to culture the organ-on-a-chip for 6-8 days; Replace the culture medium for the first chip layer (11) and the second chip layer (12), and continue culturing for 6-8 days.

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