Organ-on-chip assembly for simulating physiological barrier environment

Abstract

To provide an organ-on-chip assembly for simulating a physiological barrier environment.SOLUTION: An organ-on-chip assembly 100 includes first, second, and third microfluidic components 11,12,13 each having a microfluidic channel, wherein the second microfluidic channel is configured to receive a membrane 5, the second microfluidic component 12 is disposed between the first and third microfluidic components 11,13, and, when assembled, the microfluidic channels of the respective microfluidic components face adjacent microfluidic components and are configured such that the respective microfluidic channels are substantially aligned and connected.SELECTED DRAWING: Figure 1B

Description

[Technical Field]

[0001] The present disclosure relates to the fields of microfluidic systems and biomedical technology, and in particular to organs-on-chips and methods for simulating physiological barrier environments. [Background technology]

[0002] The brain is one of the most important and complex organs in the human body, characterized by high metabolic demands and inherent vulnerabilities. Neurological disorders such as ischemic stroke, epilepsy, and Alzheimer's disease significantly impact patients' quality of life. Effective treatment of these diseases is often hindered by the presence of the blood-brain barrier (BBB), a selective permeability barrier that limits the access of many therapeutic agents to brain tissue.

[0003] The BBB protects the brain from harmful substances in the blood while allowing essential nutrients and gases to pass through. However, this protection complicates the delivery of drugs intended to treat central nervous system (CNS) disorders. Many therapeutic agents, including large molecules and certain small molecules, cannot easily cross the BBB, resulting in insufficient drug concentrations in the brain and suboptimal therapeutic outcomes.

[0004] In vitro models simulating the BBB environment have become essential tools for the development of therapeutics targeting the central nervous system. These models allow for the investigation of drug permeability and transport mechanisms across the BBB, facilitating the screening and optimization of potential therapeutics. Typically, various brain cell types, including endothelial cells, pericytes, and astrocytes, are co-cultured on microfluidic platforms or membrane-based systems.

[0005] The development of such in vitro models is crucial for advancing our understanding of drug transport across the BBB and improving therapies for neurological disorders. By accurately reproducing the BBB in a laboratory environment, these models can accelerate the development of new central nervous system therapies and improve the quality of life for patients with debilitating brain diseases. Summary of the Invention

[0006] According to an exemplary embodiment of the present disclosure, an Organ Chip assembly includes a first microfluidic component having a first microfluidic channel, a second microfluidic component having a second microfluidic channel, the second microfluidic channel configured to receive a membrane, and a third microfluidic component having a third microfluidic channel, the first, second, and third microfluidic components being configured to be combined together such that, when combined, the first microfluidic channel of the first microfluidic component has a first portion facing the second microfluidic component and configured to substantially align and connect with the second microfluidic channel of the second microfluidic component, and the third microfluidic component has a second portion facing the second microfluidic component and configured to substantially align and connect with the second microfluidic channel of the second microfluidic component.

[0007] According to another exemplary embodiment of the present disclosure, an Organ Chip assembly includes a microfluidic device and a housing. The microfluidic device includes an upper microfluidic channel, a lower microfluidic channel, and a middle microfluidic channel between the upper and lower microfluidic channels and configured to be in fluid communication with the upper and lower microfluidic channels. The middle microfluidic channel is configured to receive a membrane. When the membrane is received in the middle microfluidic channel, a first fluid in the upper microfluidic channel flows over an upper surface of the membrane, and a second fluid in the lower microfluidic channel flows over a lower surface of the membrane. The housing is configured to secure and receive the microfluidic device.

[0008] According to another exemplary embodiment of the present disclosure, a method for simulating a physiological barrier environment includes providing a membrane, wherein a first surface and a second surface opposite to the first surface are both filled with cells; providing a microfluidic device having an upper microfluidic channel, a middle microfluidic channel, and a lower microfluidic channel; disposing the membrane within the middle microfluidic channel; supplying a first fluid to the upper microfluidic channel, wherein the first fluid immerses cells on the first surface of the membrane; and supplying a second fluid to the lower microfluidic channel, wherein the second fluid immerses cells on the second surface of the membrane.

[0009] To facilitate understanding of the present disclosure, the following embodiments are provided in conjunction with illustrations; however, the accompanying drawings are provided for reference and explanation purposes only and are not intended to limit the scope of the present disclosure. [Brief explanation of the drawings]

[0010] [Figure 1A] FIG. 1A is a schematic perspective view of an Organ Tip assembly according to an embodiment of the present disclosure. [Figure 1B]FIG. 1B is an exploded perspective view of an Organ Tip assembly according to an embodiment of the present disclosure. [Figure 2A] FIG. 2A is an exploded perspective view of a microfluidic device of an Organ Chip assembly according to an embodiment of the present disclosure. [Figure 2B] FIG. 2B is another exploded perspective view of a microfluidic device of an Organ Chip assembly according to an embodiment of the present disclosure. [Figure 3] FIG. 3 is an exploded perspective view of the housing and probe apparatus of the Organ Tip assembly according to an embodiment of the present disclosure. [Figure 4A] FIG. 4A illustrates a method for simulating a physiological barrier environment according to an embodiment of the present disclosure. [Figure 4B] FIG. 4B illustrates a method for simulating a physiological barrier environment according to an embodiment of the present disclosure. [Figure 4C] FIG. 4C illustrates a method for simulating a physiological barrier environment according to an embodiment of the present disclosure. [Figure 4D] FIG. 4D illustrates a method for simulating a physiological barrier environment according to an embodiment of the present disclosure. [Figure 4E] FIG. 4E illustrates a method for simulating a physiological barrier environment according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0011] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to illustrate certain aspects of the present disclosure. Of course, these are merely illustrative and not intended to be limiting. For example, in the following description, "forming a first feature above or on a second feature" may include embodiments in which the first and second features are formed or arranged in direct contact with each other, and may also include embodiments in which an additional feature is formed or arranged between the first and second features such that the first and second features are not in direct contact with each other. Additionally, the present disclosure may repeat reference numerals and / or letters in various examples. This repetition is for simplicity and clarity and does not, in itself, dictate a relationship between the various embodiments and / or configurations discussed.

[0012] Spatially relative terms such as "beneath," "below," "above," "over," "on," "upper," "lower," "left," "right," "vertical," "horizontal," "side," and the like may be used herein to describe the relationship of one element or feature to other element(s) or feature(s) as illustrated in the figures for ease of description. Spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation shown in the figures. The device may be in other orientations (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein will be interpreted accordingly. When an element is referred to as "connected to" or "coupled to" another element, it should be understood that the element may be directly connected or coupled to the other element, or that intervening elements may be present.

[0013] This disclosure provides a new type of sheet-like, cell-integrated microfluidic chip that can simulate the physiological environments of the vascular system, respiratory system, and / or blood-brain barrier. This is intended for research into the absorption mechanisms of targeted drugs (e.g., drugs targeted to organs, tissues, or the brain), thereby significantly reducing the need for large-scale animal experiments in drug development. By modeling the dynamics of fluid flow and the forces acting on vascular walls, phenomena such as wall deformation, arterial compliance, and pressure wave propagation in the cardiovascular system can be studied. These models will enable a better understanding of vascular characteristics in the human body and the development of more personalized and effective diagnostic tools and treatment strategies.

[0014] FIG. 1A is a schematic perspective view of an Organ Chip assembly 100 according to an embodiment of the present disclosure. FIG. 1B is an exploded perspective view of an Organ Chip assembly 100 according to an embodiment of the present disclosure. With reference to FIGS. 1A and 1B, the Organ Chip assembly 100 can include a microfluidic device 1, a housing 2, and a probe device 3. In some embodiments of the present disclosure, the Organ Chip assembly 100 is configured to simulate a physiological barrier environment. In some embodiments of the present disclosure, the physiological barrier environment includes a blood-brain barrier (BBB) ​​environment.

[0015] The microfluidic device 1 can have an upper microfluidic component 11, a middle microfluidic component 12, and a lower microfluidic component 13. As shown in FIG. 1B , a membrane 5 can be attached to the middle microfluidic component 12, sandwiching the middle microfluidic component 12 from above and below. The upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 can be stacked and combined to form the microfluidic device 1, with the middle microfluidic component 12 positioned between the upper microfluidic component 11 and the lower microfluidic component 13. In some embodiments of the present disclosure, the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 can be made primarily from polydimethylsiloxane (PDMS), a material known for its excellent biocompatibility and moldability. Therefore, when the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are stacked and combined to form the microfluidic device 1, they can exhibit good sealing properties among themselves. However, they may not be fixed in place, and movement may occur between them. The housing 2 is therefore required to secure the microfluidic device 1 formed by stacking and combining the upper microfluidic component 11, the middle microfluidic component 12 and the lower microfluidic component 13 together.

[0016] The microfluidic device 1 can have an upper microfluidic component 11, a middle microfluidic component 12, and a lower microfluidic component 13. As shown in Figure 1B, a membrane 5 can be attached to the middle microfluidic component 12, with the upper microfluidic component 11 and the lower microfluidic component 13 sandwiching the middle microfluidic component 12 from above and below to form an aggregate unit. In this configuration, the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are stacked together to form the microfluidic device 1, with the middle microfluidic component 12 positioned between the upper microfluidic component 11 and the lower microfluidic component 13.

[0017] In some embodiments of the present disclosure, the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 can be made primarily from polydimethylsiloxane (PDMS), a material known for its excellent biocompatibility and moldability. PDMS is a silicon-based organic polymer widely used in biomedical and microfluidic applications due to its flexibility, optical transparency, and ease of fabrication. PDMS can be used to create complex microchannel structures that can accurately replicate physiological conditions, which are essential for studying cell behavior and drug interactions in controlled environments.

[0018] Therefore, when the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are stacked and combined to form the microfluidic device 1, they exhibit good sealing properties among themselves. Such good sealing properties are important for preventing fluid leakage, which is essential for maintaining the integrity of the simulated physiological environment within the microfluidic device. However, due to the inherent flexibility of PDMS, the components may not be fixed in place, leading to potential movement between the components. Such movement may disrupt the precise alignment and function of the microfluidic channels and affect experimental results.

[0019] Therefore, the housing 2 is required to secure the microfluidic device 1, which is formed by stacking and assembling the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13. The housing provides structural stability and ensures that the components remain in the correct position during operation. This stability is crucial for conducting accurate and reproducible experiments, as even slight misalignment of the microfluidic components can affect the flow dynamics within the device and the interactions between different cell types. The housing also helps protect the microfluidic device from external physical damage and contamination, further improving the reliability and lifespan of the device.

[0020] Referring to Figure 1B, the housing 2 can have a main body 21 and a cover 22. The main body 21 of the housing 2 can have an interior space 210 designed to securely house the microfluidic device 1. Once the microfluidic device 1 is placed within the interior space 210, fasteners 25 can be used to fasten the cover 22 and securely hold the device in place. This secure encapsulation is crucial for maintaining the integrity of the microfluidic system and preventing leakage or slippage during experimental procedures. The housing 2 is typically made of titanium alloys, polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), polyethylene terephthalate (PET), acrylonitrile butadiene styrene (ABS), or polyolefins, selected for their high biocompatibility and durability. These materials are widely used in biomedical applications due to their strength, corrosion resistance, and compatibility with biological tissue.

[0021] The housing 2 can also include multiple fluid connectors 23. These connectors are configured to introduce external fluids into the microfluidic device 1 and to introduce fluids from within the microfluidic device 1 to the outside. Precise control of fluid flow is essential for replicating the dynamic environment of the BBB. By introducing various fluids, such as cell culture media, drugs, and other reagents, researchers can simulate various biological scenarios and study their effects on cells within the microfluidic device. The ability to control fluid dynamics is crucial for maintaining a desirable microenvironment and ensuring reproducible experimental results.

[0022] 1A and 1B, the probe device 3 is configured to fit over the cover 22 of the housing 2. The probe device may include a probe box 31, a top cover 32 configured to cover the probe box 31, and a plurality of electrode probes 33 and 35 extending from the probe box 31. When the probe device 3 is attached to the cover 22 of the housing 2, the electrode probes 33 and 35 can extend into the microfluidic device 1. This allows the probe device 3 to perform signal measurements on the fluid within the microfluidic device 1. Examples of signal measurements include, but are not limited to, electrical signals (such as transepithelial electrical resistance (TEER)), optical signals, thermal signals, and vibration signal measurements.

[0023] In embodiments, TEER measurements are a key indicator of barrier function, allowing for real-time monitoring of electrical resistance across cell layers. This is particularly important for BBB research, as it helps understand how different compounds affect the barrier's permeability and integrity. The probe device 3 enables accurate measurements, facilitating detailed studies of drug delivery, transport mechanisms, and the impact of various pathologies on the BBB. This capability is essential for developing new therapeutic strategies and improving drug formulations targeting the central nervous system.

[0024] FIG. 2A is an exploded perspective view of the microfluidic device 1 of the Organ Chip assembly 100 according to an embodiment of the present disclosure. FIG. 2B is another exploded perspective view of the microfluidic device 1 of the Organ Chip assembly 100 according to an embodiment of the present disclosure. As described above, the microfluidic device 1 can have an upper microfluidic component 11, a middle microfluidic component 12, and a lower microfluidic component 13. Furthermore, the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 can be stacked and combined to form the microfluidic device 1. The upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 can be primarily made of polydimethylsiloxane (PDMS). PDMS exhibits excellent biocompatibility, making it suitable for medical and bioengineering applications. Polydimethylsiloxane (PDMS) has high elasticity and flexibility and can be easily processed into various shapes and structures. PDMS also has excellent gas permeability, allowing for efficient exchange of oxygen and carbon dioxide, making it ideal for cell culture and microfluidic devices. Its transparency allows for easy observation and imaging, and its chemical inertness, low toxicity, and compatibility with most chemical and biological substances further enhance its versatility for a variety of applications.

[0025] 2A and 2B, the upper microfluidic component 11 may have a lower surface 111, which may face the intermediate microfluidic component 12 when the upper microfluidic component 11, the intermediate microfluidic component 12, and the lower microfluidic component 13 are assembled together. The upper microfluidic component 11 may have an upper microfluidic channel 110 formed in the lower surface 111. That is, the upper microfluidic channel 110 may also face the intermediate microfluidic component 12 when the upper microfluidic component 11, the intermediate microfluidic component 12, and the lower microfluidic component 13 are assembled together. The upper microfluidic channel 110 may have a fluid inlet 1102 and a fluid outlet 1104. The fluid inlet 1102 may be located on one side 113 of the upper microfluidic component 11, and the fluid outlet 1104 may be located on the opposite side 115 of the upper microfluidic component 11. That is, fluid can be introduced into the upper microfluidic channel 110 through a fluid inlet 1102 from a side 113 of the upper microfluidic component 11 and can be discharged from a side 115 of the upper microfluidic component 11 through a fluid outlet 1104 .

[0026] The upper microfluidic channel 110 may have a circular recess 1101. The circular recess 1101 may be located substantially in the middle of the fluid path formed by the upper microfluidic channel 110. The depth of the circular recess 1101 may be substantially deeper than the depth of other portions of the upper microfluidic channel 110. Furthermore, the circular recess 1101 may substantially match the shape of the membrane 5 attached to the middle microfluidic component 12. Therefore, when the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are assembled together, the circular recess 1101 may cover the upper surface of the membrane 5 attached to the middle microfluidic component 12, and fluid passing through the circular recess 1101 of the upper microfluidic channel 110 may flow over the upper surface of the membrane 5.

[0027] Furthermore, the upper microfluidic channel 110 can include multiple barriers 1107. Each of the barriers 1107 can extend substantially along the flow direction of the fluid flowing through the upper microfluidic channel 110. Furthermore, the barriers 1107 can be positioned adjacent to the circular recess 1101. The barriers 1107 are configured to disrupt the flow of the fluid flowing through the upper microfluidic channel 110. That is, the barriers 1107 can simulate the shear stress and pulsatile micromotion of the cerebral blood vessel wall. The barriers 1107 can be positioned adjacent to the circular recess 1101, and the barriers 1107 can create flow conditions for the fluid flowing through the recess 1101 that are similar to the shear stress and pulsatile flow experienced by endothelial cells in cerebral blood vessels. In microfluidic systems, the design and structural elements within the channel are crucial for simulating physiological conditions. By incorporating barriers such as baffles or grids, it is possible to create flow conditions similar to the shear stress and pulsatile flow experienced by endothelial cells in cerebral blood vessels. Shear stress is generated when barriers disrupt laminar flow, creating regions of varying flow velocity, localized high shear regions, and more uniform shear stress distribution. Furthermore, barriers induce pulsatile flow by causing periodic flow oscillations, pressure fluctuations, and flow reversals, simulating the dynamic environment of cardiac-driven blood flow. These characteristics are essential for accurately mimicking the mechanical signals and conditions that influence cell behavior in vivo.

[0028] Additionally, the upper microfluidic component may further have a fluid inlet 1103 and a fluid outlet 1105. The fluid inlet 1103 may be located on a side surface 115 of the upper microfluidic component 11, while the fluid outlet 1105 may be located on an opposite side surface 113 of the upper microfluidic component 11. The fluid inlet 1103 is configured to introduce fluid into the lower microfluidic channel 130 of the lower microfluidic component 13, and the fluid outlet 1105 is configured to discharge fluid from the channel 130. Further details regarding the structure of the lower microfluidic channel 130 of the lower microfluidic component 13 are provided later in this specification.

[0029] Furthermore, the upper microfluidic component 11 may have through vias 1121 and 1122 formed on the upper surface 112 of the upper microfluidic component 11 and penetrating the upper microfluidic component 11. The through via 1121 may be in fluid communication with the upper microfluidic channel 110 of the upper microfluidic component 11. In some embodiments of the present disclosure, the through via 1121 is connected to the fluid outlet 1104. The through via 1122 may be in fluid communication with the lower microfluidic channel 130 of the lower microfluidic component 13. In some embodiments of the present disclosure, the through via 1122 is connected to the fluid inlet 1103. That is, the electrode probes 33 and 35 of the probe device 3 may extend into the upper microfluidic channel 110 and the lower microfluidic channel 130 through the through vias 1121 and 1122.

[0030] The middle microfluidic component 12 can have a middle microfluidic channel 120. The middle microfluidic channel 120 is configured to be in fluid communication with the upper microfluidic channel 110 of the upper microfluidic component 11 and the lower microfluidic channel 130 of the lower microfluidic component 13. In some embodiments of the present disclosure, the middle microfluidic channel 120 has a circular opening that penetrates the middle microfluidic component 12. The opening is configured to receive a membrane 5. That is, the membrane 5 can be attached to the middle microfluidic channel 120 of the middle microfluidic component 12. When the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are stacked and combined, the circular recess 1101 of the upper microfluidic channel 110 of the upper microfluidic component 11 can align with the opening of the middle microfluidic channel 120 of the middle microfluidic component 12 and can mate with the membrane 5 received in the opening of the middle microfluidic channel 120 of the middle microfluidic component 12. Similarly, the circular recess 1301 of the lower microfluidic channel 130 of the lower microfluidic component 13 aligns with the opening of the intermediate microfluidic channel 120 of the intermediate microfluidic component 12, and can likewise mate with the membrane 5 received in the opening of the intermediate microfluidic channel 120 of the intermediate microfluidic component 12. Further details regarding the structure of the lower microfluidic channel 130 of the lower microfluidic component 13 will be described below.

[0031] Furthermore, the middle microfluidic component 12 can have through-holes 121 and 122. The through-hole 121 is configured to align with and / or connect to a fluid outlet 1105 disposed on the side surface 113 of the upper microfluidic component 11. The through-hole 122 is configured to align with and / or connect to a fluid inlet 1103 disposed on the side surface 115 of the upper microfluidic component 11. When the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are stacked and combined, the through-hole 121 can be in fluid communication with the fluid outlet 1105, and the through-hole 122 can be in fluid communication with the fluid inlet 1103.

[0032] 2A and 2B, the lower microfluidic component 13 may have an upper surface 131, which may face the intermediate microfluidic component 12 when the upper microfluidic component 11, the intermediate microfluidic component 12, and the lower microfluidic component 13 are assembled together. The lower microfluidic component 13 may have a lower microfluidic channel 130 formed in the upper surface 131. That is, the lower microfluidic channel 130 may also face the intermediate microfluidic component 12 when the upper microfluidic component 11, the intermediate microfluidic component 12, and the lower microfluidic component 13 are assembled together. The lower microfluidic channel 130 may have ends 1303 and 1305. The end 1303 of the lower microfluidic channel 130 is configured to align with the through-hole 121 of the intermediate microfluidic component 12. The end 1305 of the lower microfluidic channel 130 is configured to align with the through-hole 122 of the intermediate microfluidic component 12. When the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are assembled together, the end 1303 of the lower microfluidic channel 130 can be in fluid communication with the through-hole 121 of the middle microfluidic component 12, and the end 1305 of the lower microfluidic channel 130 can be in fluid communication with the through-hole 122 of the middle microfluidic component 12. Furthermore, as described above, the through-hole 122 can be in fluid communication with the fluid inlet 1103, and the through-hole 121 can be in fluid communication with the fluid outlet 1105. That is, fluid can be introduced into the lower microfluidic channel 130 through the fluid inlet 1103 on the side surface 115 of the upper microfluidic component 11 and the through-hole 122 of the middle microfluidic component 12, and can be discharged from the side surface 113 of the upper microfluidic component 11 through the through-hole 121 of the middle microfluidic component 12 and the fluid outlet 1105 on the side surface 113 of the upper microfluidic component 11.Furthermore, since the end 1305 of the lower microfluidic channel 130, the through-hole 122 and the fluid inlet 1103 are fluidly connected to each other, the electrode probe 35 can extend through the through-via 1122 and the through-hole 122 to the end 1305 of the lower microfluidic channel 130.

[0033] The lower microfluidic channel 130 may have a circular recess 1301. The circular recess 1301 may be located substantially in the middle of the fluid path formed by the lower microfluidic channel 130. The depth of the circular recess 1301 may be substantially deeper than the depth of other portions of the lower microfluidic channel 130. Furthermore, the circular recess 1301 may substantially conform to the shape of the membrane 5 attached to the middle microfluidic component 12. Therefore, when the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are assembled together, the circular recess 1301 may cover the lower surface of the membrane 5 attached to the middle microfluidic component 12, and fluid passing through the circular recess 1301 of the lower microfluidic channel 130 may flow over the lower surface of the membrane 5.

[0034] Furthermore, the lower microfluidic channel 130 can include multiple barriers 1307. Each of the barriers 1307 can extend substantially along the flow direction of the fluid flowing through the lower microfluidic channel 130. Furthermore, the barriers 1307 can be positioned adjacent to the circular recess 1301. The barriers 1307 are configured to disrupt the flow of the fluid flowing through the lower microfluidic channel 130. That is, the barriers 1307 can simulate the shear stress and pulsatile micromotion of the cerebral blood vessel wall. In microfluidic systems, the design and structural elements within the channel are crucial for simulating physiological conditions. Because the barriers 1307 can be positioned adjacent to the circular recess 1301, the barriers 1307 can create flow conditions for the fluid flowing through the recess 1301 that are similar to the shear stress and pulsatile flow experienced by endothelial cells in cerebral blood vessels. By incorporating barriers such as baffles or gratings, it is possible to create flow conditions similar to the shear stress and pulsatile flow experienced by endothelial cells in cerebral blood vessels. Shear stress is generated when barriers disrupt laminar flow, creating regions of varying flow velocity, localized high shear regions, and more uniform shear stress distribution. Furthermore, barriers induce pulsatile flow by causing periodic flow oscillations, pressure fluctuations, and flow reversals, simulating the dynamic environment of cardiac-driven blood flow. These characteristics are essential for accurately mimicking the mechanical signals and conditions that influence cell behavior in vivo.

[0035] FIG. 3 is an exploded perspective view of the housing 2 and probe device 3 of the Organ Chip assembly 100 according to an embodiment of the present disclosure. As shown in FIG. 3, the housing 2 can include a main body 21 and a cover 22. The main body 21 of the housing 2 can have an interior space 210 configured to accommodate the microfluidic device 1 formed by stacking and assembling the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13. As described above, the middle microfluidic component 12 and the lower microfluidic component 13 can be made primarily from polydimethylsiloxane (PDMS), a material known for its excellent biocompatibility and moldability. As a result, when the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are stacked and assemblable to form the microfluidic device 1, they exhibit good sealing properties. However, without proper fixation, there is a risk of relative movement between these components. The housing 2 is therefore configured to secure the microfluidic device 1, ensuring that the stacked and combined top, middle and bottom microfluidic components 11, 12, 13 remain fixed in place, providing further stability and preventing potential displacement.

[0036] In this embodiment, the housing 2 is primarily made of a titanium alloy. Titanium alloys have excellent mechanical properties and biocompatibility, making them suitable for medical and bioengineering applications. Their high strength-to-weight ratio allows for lightweight construction while maintaining the structural integrity critical to platform design. In addition, titanium alloys exhibit remarkable corrosion resistance, allowing them to withstand humid environments and extended use within biological systems without degradation, thereby enhancing the durability and reliability of the housing 2. Furthermore, titanium alloys' low coefficient of thermal expansion and excellent thermal stability ensure dimensional stability under temperature changes, which is essential for accurate operation and measurement in microfluidic systems. In summary, the use of titanium alloys not only provides mechanical strength and stability, but also ensures biocompatibility and long-term durability.

[0037] In some embodiments of the present disclosure, housing 2 is equipped with a temperature-controlled water channel within body 21 that can be connected to an external water circuit for temperature regulation, thereby allowing Organ Chip assembly 100 to maintain a stable temperature at a set physiological temperature or any other desired temperature.

[0038] The body 21 of the housing 2 may have a window 211. The cover 22 of the housing 2 may have a window 221. When the microfluidic device 1 formed by stacking and assembling the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 is enclosed in the housing 2, the windows 211 and 221 may be substantially aligned with the membrane 5 attached to the middle microfluidic component 12 of the microfluidic device 1. Because the PDMS used in the fabrication of the microfluidic device 1 may have transparent properties, a user can observe the membrane 5 attached to the middle microfluidic component 12 of the microfluidic device 1 through the windows 211 and 221 when using the Organ Chip assembly 100.

[0039] The housing 2 may have four fluid connectors 23. These fluid connectors 23 are configured to connect the fluid inlets 1102 and 1103 and the fluid outlets 1104 and 1105 of the microfluidic device 1. In some embodiments of the present disclosure, the connectors 23 may have Luer connectors. The Luer connectors are configured to connect an external tubing system containing the input and output of cerebral blood and cerebral fluid. In some embodiments, the connector 23 connected to the fluid inlet 1102 can function as the cerebral fluid input, and the connector 23 connected to the fluid outlet 1104 can function as the cerebral fluid output. This configuration allows cerebral fluid to flow through the upper microfluidic channel 110. In some embodiments, the connector 23 connected to the fluid inlet 1103 can function as the cerebral blood input, and the connector 23 connected to the fluid outlet 1105 can function as the cerebral blood output. This configuration allows cerebral blood to flow through the lower microfluidic channel 110. The main function of the Luer connectors is to ensure a stable connection between the tubing inside and outside the microfluidic system.

[0040] After the microfluidic device 1 is installed in the interior space 210 of the main body 21 of the housing 2, the cover 22 is secured in place using fasteners 25. These fasteners 25 not only provide a secure fit, but also maintain the integrity of the upper, middle, and lower microfluidic components 11, 12, and 13, preventing potential leakage or displacement during operation. Secure fit of the cover 22 allows the Organ Chip assembly 100 to operate reliably under stable conditions, which are essential for accurate experimental results and reliable performance in a variety of biomedical applications.

[0041] The probe apparatus 3 may have a probe box 31, an upper cover 32 configured to cover the probe box 31, and a plurality of electrode probes 33 and 35 extending from the probe box 31. The probe apparatus 3 may be received in a recess 222 formed in an upper surface 220 of the cover 22 of the housing 2. The recess 222 may have through vias 2221 and 2222, where the through via 2221 may be substantially aligned with the through via 1121 of the upper microfluidic component 11 of the microfluidic device 1, and the through via 2222 may be substantially aligned with the through via 1122 of the upper microfluidic component 11 of the microfluidic device 1. When the probe apparatus 3 is attached to the housing 2 and received in the recess 222 of the cover 22, the electrode probes 33 may pass through the through vias 2221 of the recess 222 of the cover 22 and the through via 1121 of the upper microfluidic component 11 of the microfluidic device 1, and extend into the upper microfluidic channel 110. The electrode probe 35 can extend through the through via 2222 in the recess 222 of the cover 22, the through via 1122 in the upper microfluidic component 11 of the microfluidic device 1, and the through hole 122 in the middle microfluidic component 12 into the lower microfluidic channel 110.

[0042] 4A, 4B, 4C, 4D, and 4E illustrate a method for simulating a physiological barrier environment according to an embodiment of the present disclosure. Referring to FIG. 4A, a membrane 5 having cell groups 510 and 520 is provided. As shown in FIG. 4A, cell group 510 is cultured on an upper surface 51 of the membrane 5, and cell group 520 is cultured on a lower surface 52 of the membrane 5. In some embodiments of the present disclosure, cell groups 510 and 520 may include endothelial cells, pericytes, and astrocytes. That is, endothelial cells, pericytes, and astrocytes may be co-cultured on the upper and lower surfaces 51 and 52 of the membrane 5, thereby establishing a tissue structure similar to that of cerebral blood vessels. In other words, the membrane 5 has a simulated blood-brain barrier (BBB) ​​environment.

[0043] 4B , the membrane 5 is attached to the middle microfluidic channel 120 of the middle microfluidic component 12, and the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are stacked and assembled to form the microfluidic device 1. The middle microfluidic component 12 is disposed between the upper microfluidic component 11 and the lower microfluidic component 13. The upper microfluidic channel 110 faces the middle microfluidic component 12, and the recess 1101 of the upper microfluidic channel 110 is aligned with the middle microfluidic channel 120 and covers the upper surface 51 of the membrane 5. The lower microfluidic channel 130 faces the middle microfluidic component 12, and the recess 1301 of the lower microfluidic channel 130 is aligned with the middle microfluidic channel 120 and covers the lower surface 52 of the membrane 5.

[0044] The upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 are primarily made of polydimethylsiloxane (PDMS), which provides good sealing when stacked together but is difficult to fix in place. Therefore, the upper microfluidic component 11, the middle microfluidic component 12, and the lower microfluidic component 13 can be sealed in the housing 2 to securely fix them in place. After the combined upper, middle, and lower microfluidic components 11, 12, and 13 are sealed in the housing 2, an external water circuit can be connected to the temperature-controlled water channel in the main body 21 of the housing 2 to maintain the internal temperature of the housing 2 at 37 °C or a desired temperature.

[0045] 4C , a fluid 119 is introduced into the upper microfluidic channel 110, and a fluid 129 is introduced into the lower microfluidic channel 130. In some embodiments of the present disclosure, the fluid 119 includes cerebral fluid, and the fluid 129 includes cerebral blood. The fluid 119 enters the upper microfluidic channel 110 through a fluid inlet 1102 and exits the upper microfluidic channel 110 through a fluid outlet 1104. The fluid 119 flows over the upper surface 51 of the membrane 5. A group of cells 510 on the upper surface 51 of the membrane 5 is immersed in the first fluid 119.

[0046] 4C , the fluid 129 flows into the lower microfluidic channel 130 through the fluid inlet 1103 and the through-hole 122, and flows out of the lower microfluidic channel 130 through the through-hole 121 and the fluid outlet 1104. The fluid 129 flows on the lower surface 52 of the membrane 5. The cell group 520 on the lower surface 52 of the membrane 5 is immersed in the first fluid 129. Furthermore, as shown in FIG. 4C , the flow direction of the fluid 119 in the upper microfluidic channel 110 is substantially opposite to the flow direction of the fluid 129 in the lower microfluidic channel 130.

[0047] The fluid inlet 1102 and fluid outlet 1104 for introducing the flow of fluid 119 can be connected to the fluid connector 23 of the housing 2, and the fluid inlet 1103 and fluid outlet 1105 for introducing the flow of fluid 129 can be connected to the fluid connector 23 of the housing 2. The fluid connector 23 can have a luer connector. The luer connector is configured to ensure a stable connection between the internal channel and the external tubing.

[0048] Referring to FIG. 4D , a test drug is added to the upper microfluidic channel 110 and / or the lower microfluidic channel 130, and the absorption and permeation of the test drug in the blood-brain barrier environment simulated by the membrane 5 is observed. As shown in FIG. 4D , when fluid 119 flows through the barrier 1107, the barrier 1107 can disturb the flow of fluid 119, and when fluid 129 flows through the barrier 1307, the barrier 1307 can disturb the flow of fluid 129. The placement of the barrier 1107 in the upper microfluidic channel 110 and the barrier 1307 in the lower microfluidic channel 130 is intended to further simulate and generate shear stress and pulsatile motion similar to those on the wall of a cerebral blood vessel. In particular, as shown in FIG. 4D , the barrier 1107 in the upper microfluidic channel 110 can create flow conditions for fluid 119 flowing over the upper surface 51 of the membrane 5 similar to the shear stress and pulsatile flow experienced by endothelial cells in a cerebral blood vessel. Furthermore, the barrier 1307 of the lower microfluidic channel 130 can create flow conditions for the fluid 129 flowing over the lower surface 52 of the membrane 5 similar to the shear stress and pulsatile flow experienced by endothelial cells in cerebral blood vessels.

[0049] Referring to Figure 4E, real-time monitoring of transepithelial electrical resistance (TEER) is performed. As shown in Figure 4E, electrode probe 33 extends into upper microfluidic channel 110 through through-via 1121, and electrode probe 35 extends into lower microfluidic channel 130 through through-hole 122 and through-via 1122. Therefore, the electrical resistance of membrane 5 can be monitored in real time.

[0050] Additionally, housing 2 has windows 211 and 221 so that Organ Chip Assembly 100 can be placed and observed under optical microscopes (both visible light and fluorescent microscopes).

[0051] This new microfluidic device offers significant advantages for the development of brain-targeted drugs, primarily by reducing reliance on animal testing. The technology significantly reduces the number of animals required for drug development and addresses the ethical concerns and logistical challenges associated with animal testing. By providing an accurate in vitro model of the blood-brain barrier (BBB), researchers can conduct comprehensive drug testing without extensive use of animal models.

[0052] Another key advantage is the device's real-time monitoring capability. Drug absorption mechanisms in the brain can be continuously observed, providing immediate and accurate data on drug efficacy and permeability. This real-time feedback is crucial for optimizing drug formulations and understanding interactions with the BBB, significantly improving the drug development process.

[0053] Furthermore, this device accurately simulates the physiological environment of the BBB. By reproducing the cerebral vascular conditions, it provides a highly realistic experimental platform that closely resembles actual human brain tissue. This improves the predictive accuracy of in vitro drug testing, providing results that more closely reflect in vitro conditions. As a result, this device improves the reliability and validity of preclinical drug evaluation and accelerates the development of effective and safe treatments for neurological diseases.

[0054] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise.

[0055] As used herein, the terms "approximately," "substantially," "substantial," and "about" are used to describe and explain slight variations. When used in conjunction with an event or circumstance, these terms can refer not only to instances in which the event or circumstance occurs exactly, but also to instances in which the event or circumstance occurs approximately. For example, when used in conjunction with a numerical value, these terms can refer to a range of variation of the numerical value of ±10% or less, such as ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, or ±0.05%. For example, two numerical values ​​can be considered "substantially" identical or equal if the difference between the two numerical values ​​is ±10% or less of the mean value of the numerical values, such as ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, or ±0.05%. For example, "substantially" parallel can refer to a range of angular variation from 0° of ±10° or less, e.g., ±5° or less, ±4° or less, ±3° or less, ±2° or less, ±1° or less, ±0.5° or less, ±0.1° or less, or ±0.05° or less. For example, "substantially" perpendicular can refer to a range of angular variation from 90° of ±10° or less, e.g., ±5° or less, ±4° or less, ±3° or less, ±2° or less, ±1° or less, ±0.5° or less, ±0.1° or less, or ±0.05° or less.

[0056] Additionally, quantities, ratios, and other numerical values ​​may be presented herein in a range format. It will be understood that such range format is used for convenience and brevity and should be understood flexibly to include not only the numerical values ​​expressly specified as the limits of the range, but also to include all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly specified.

[0057] While the present disclosure has been described and illustrated with reference to specific embodiments thereof, these descriptions and illustrations are not intended to limit the disclosure. Those skilled in the art should understand that various changes may be made and equivalents substituted without departing from the true spirit and scope of the present disclosure, as defined by the appended claims. Illustrations may not necessarily be drawn to scale. Manufacturing processes and tolerances may result in differences between the depictions in the drawings of this disclosure and the actual device. There may be other embodiments of the present disclosure not specifically illustrated. The specification and drawings are to be considered illustrative, not restrictive. Modifications may be made to adapt particular situations, materials, mixtures, methods, or processes to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. Although methods disclosed herein are described with reference to particular operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or re-sequenced to form equivalent methods without departing from the teachings of the present disclosure. Accordingly, unless otherwise indicated herein, the order and grouping of operations is not intended to limit the disclosure.

Claims

1. a first microfluidic component having a first microfluidic channel; a second microfluidic component having a second microfluidic channel, the second microfluidic channel configured to receive a membrane; a third microfluidic component having a third microfluidic channel; the first microfluidic component, the second microfluidic component, and the third microfluidic component are configured to be combined, and the second microfluidic component is disposed between the first microfluidic component and the third microfluidic component such that, when combined, the first microfluidic channel of the first microfluidic component has a first portion facing the second microfluidic component and configured to substantially align and connect with the second microfluidic channel of the second microfluidic component, and the third microfluidic channel of the third microfluidic component has a second portion facing the second microfluidic component and configured to substantially align and connect with the second microfluidic channel of the second microfluidic component.

2. 10. The Organ Chip assembly of claim 1, wherein the first microfluidic channel of the first microfluidic component is configured to receive a first fluid and the third microfluidic channel of the third microfluidic component is configured to receive a second fluid, the first fluid being different from the second fluid.

3. 3. The Organ Chip assembly of claim 2, wherein the first microfluidic channel of the first microfluidic component has a plurality of first barriers, each of the first barriers extending substantially along the direction of flow of the first fluid, and the third microfluidic channel of the third microfluidic component has a plurality of second barriers, each of the second barriers extending substantially along the direction of flow of the second fluid.

4. 4. The Organ Chip assembly of claim 3, wherein the plurality of first barriers are disposed adjacent to the first portion of the first microfluidic channel and the plurality of second barriers are disposed adjacent to the second portion of the third microfluidic channel.

5. 10. The Organ Chip assembly of claim 1, wherein the first microfluidic component comprises a first inlet, a second inlet, a first outlet, and a second outlet; the second microfluidic component comprises a first through-hole and a second through-hole, the first inlet and the first outlet being in liquid communication with the first microfluidic channel, the first through-hole substantially aligned with the second inlet and in fluid communication with the third microfluidic channel of the third microfluidic component, and the second through-hole substantially aligned with the second outlet and in fluid communication with the third microfluidic channel of the third microfluidic component.

6. 6. The Organ Chip assembly of claim 5, wherein the first inlet and the second outlet are disposed on a first side of the first microfluidic component, and the first outlet and the second inlet are disposed on a second side of the first microfluidic component opposite the first side.

7. 6. The Organ Chip assembly of claim 5, further comprising a housing configured to enclose said first microfluidic component, said second microfluidic component, and said third microfluidic component.

8. 8. The organ tip assembly of claim 7, wherein the housing comprises four liquid connectors, each of the four liquid connectors being in fluid communication with the first inlet, the first outlet, the second inlet, and the second outlet, respectively.

9. 8. The Organ Chip assembly of Claim 7, wherein the housing comprises a first via and a second via, the first microfluidic component comprises a third via substantially aligned with the first via of the housing and connected to the first outlet, and a fourth via substantially aligned with the second via and connected to the second inlet, the first and third vias configured to allow a first electrode probe to extend into the first microfluidic channel, and the second and fourth vias configured to allow a second electrode probe to extend into the third microfluidic channel.

10. 10. The Organ Chip assembly of claim 9, further comprising a probe device having said first electrode probe and said second electrode probe, said probe device configured to fit into said housing.

11. 10. The Organ Chip Assembly of claim 1, wherein the first microfluidic component, the second microfluidic component, and the third microfluidic component comprise a polydimethylsiloxane (PDMS) material.

12. 8. The Organ Tip Assembly of claim 7, wherein the housing comprises a titanium material, polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), polyethylene terephthalate (PET), acrylonitrile butadiene styrene (ABS), or polyolefin.

13. 8. The Organ Chip Assembly of Claim 7, wherein said housing comprises at least one window aligned with said second microfluidic channel of said second microfluidic component.

14. an upper microfluidic channel; a lower microfluidic channel; an intermediate microfluidic channel located between the upper microfluidic channel and the lower microfluidic channel and configured to be in fluid communication with the upper microfluidic channel and the lower microfluidic channel; The microfluidic device, wherein the middle microfluidic channel is configured to receive a membrane, and when the membrane is received in the middle microfluidic channel, a first fluid in the upper microfluidic channel flows over an upper surface of the membrane and a second fluid in the lower microfluidic channel flows over a lower surface of the membrane.

15. 15. The microfluidic device of claim 14, wherein the upper microfluidic channel includes a portion configured to substantially conform to a shape of the membrane received in the middle microfluidic channel, and the lower microfluidic channel includes a portion configured to substantially conform to the shape of the membrane received in the middle microfluidic channel.

16. 15. The microfluidic device of claim 14, wherein the upper microfluidic channel comprises one or more first barriers configured to disrupt the flow of the first fluid in the upper microfluidic channel, and the lower microfluidic channel comprises one or more second barriers configured to disrupt the flow of the second fluid in the lower microfluidic channel.

17. providing a membrane, wherein a first surface of the membrane and a second surface opposite the first surface are both filled with cells; providing a microfluidic device having an upper microfluidic channel, a middle microfluidic channel, and a lower microfluidic channel; disposing the membrane within the intermediate microfluidic channel; supplying a first fluid into the upper microfluidic channel, the first fluid immersing cells on the first surface of the membrane; and supplying a second fluid to the lower microfluidic channel, the second fluid immersing cells on the second surface of the membrane.

18. perturbing the flow of the first fluid in the upper microfluidic channel; 20. The method of claim 17, further comprising: disturbing the flow of the second fluid in the lower microfluidic channel.

19. providing a first electrode probe in the upper microfluidic channel and a second electrode probe in the lower microfluidic channel; The method of claim 17, further comprising measuring the electrical resistance of the film.

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