Apparatus with 3D microfluidic features for in vitro culturing applications

Abstract

A three-dimensional microfluidic device is provided. The device includes an array of fluid transporting channels formed in a permeable gel. The channels allow both direct circulation flow and diffusion flow from a channel into the gel. The diffusion flow is lateral to the direction of the channel. The device also includes one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the permeable gel. The device further includes one or more types of organoids cultured in the device.

Description

APPARATUS WITH 3D MICROFLUIDIC FEATURES FOR IN VITRO CULTURING APPLICATIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 528,221, filed July 21, 2023, which application is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] This invention relates generally to in vitro culturing.BACKGROUND OF THE INVENTION

[0003] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0004] The blood-brain barrier (BBB) refers to the biochemical, metabolic and protective interface between the central nervous system (CNS) and the capillary blood vessels within it. It prevents irregular changes in the environment, like neurotoxic components, from reaching the CNS but allows nutrition and oxygen to go through to support the CNS. The CNS also releases communication chemicals into the vascular system through the BBB. Thus, the study of the BBB is of great interest to scientists and clinicians as the integrity of the BBB is essential for the functionality and health of the CNS. BBB dysfunctions are implicated in a collection of neurological disorders, e.g., Alzheimer’s disease. Studies in drug delivery to the CNS also consider the BBB meaningful as the drug needs to go through the BBB to reach the CNS.

[0005] The BBB formation requires strict conditions with the presence of continuous blood flow that allows the constant inducing and signal interaction from the CNS. As a result, previous studies on the BBB were usually carried out on live animal subjects where the BBB was already formed or relatively easy to be formed under suitable environment. This brings uncertainty to the study and limits the varieties of BBB to be studied, due to limited control of the animal subject breeding process during which the desired traits must be expressed. The demand for in vitro reconstruction of BBB has then emerged, which requires the co-culturing of brain and blood vessel organoids with desired traits expressed during the inducing of stem cells. Unfortunately, this has not been realized yet at least partially due to the difficulty in reproducing the circulation environment required for BBB to form at a size scale that suits organoids. Concepts of in vitro modelingprocedures at the cellular scale have been conceived in both 2D and 3D. Previous works have utilized such concepts and demonstrated in vitro culturing of blood vessel cells in microfluidic devices under continuous flows, inducing them to form vessel tissues. However, these devices, typically planar in nature, have a majority of the flow going horizontally, with only minor vertical flow motion occurring, e.g., inside porous foams or through leak holes on a substrate, giving a height / volume as well as flow configurations that are not suitable for organoids with typical sizes of a few mm or more. Flows that cover the entire size of the organoids are essential as they provide the shear force to stimulate the formation of BBB. Embedded 3D printing has been used to form channels by printing sacrificial ink that was later removed, but the process relies on the self-healing capability of organoids due to its invasive nature that requires the positioning of the printing nozzle inside the tissue volume, limiting its suitable range of applications. Therefore, a need still exists for a method of creating an artificial BBB model that is suitable for testing organoid-sized materials while emulating a continuous and controllable blood flow pattern.SUMMARY OF THE INVENTION

[0006] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0007] Many of the drawbacks and limitations stated above can be resolved by creating novel and advanced interplays of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, in a manner that affordably, effectively, conveniently, intelligently, or reliably brings sensing technology into proximity with biofluid and analytes.

[0010] In one aspect of the present invention, a three-dimensional microfluidic device is provided. The device enables in vitro emulation of a blood brain barrier environment of a human body. The device includes an array of fluid transporting channels formed in a permeable gel. The channels allow both direct circulation flow and diffusion flow from a channel into the gel. The diffusion flow is lateral to the direction of the channel. Also, the channels are at the size scale of whole organoids. The device also includes one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the permeable gel. The device further includes one or more types of organoids cultured in the device. The device is capable of emulating a microscopic and macroscopic blood flow pattern in the body and enables the study of how changes in the blood flow pattern affect the development of organoids.

[0011] “Fluid movement devices” include devices suitable for moving fluid through the device of the present invention. In various embodiments, the fluid movement devices are one or more fluid pumps, vacuum pumps, pressure blowers, or fans.

[0012] In one embodiment, the organoids include both brain organoids and blood vessel organoids and the brain organoids and blood vessel organoids are co-cultured together. In another embodiment, the fluid movement devices are pumps. In one embodiment, the device further includes a substrate with one or more embedded channels. Each embedded channel is in fluid communication with the fluid transporting channels. The gel surrounds a second gel material that includes at least one volume for housing and culturing one or more organoids. Also, the device includes a suction device to remove fluid from the device. In one embodiment, the blood vessel organoid responds to blood flow pattern when growing and thus the growth can be affected by changing the flow parameters.

[0013] In another embodiment, the permeable gel and second gel material are supported on one or more diaphragms. The diaphragms have a plurality of holes and the holes are matched to the locations of the fluid transporting channels. In one embodiment, the fluid transporting channels have a diameter. Also, the holes in the one or more diaphragms have a diameter that is the same as the diameter of the fluid transporting channels.

[0014] In another embodiment, the holes in the one or more diaphragms have a diameter in the range of 1 pm to 1000 pm. In one embodiment, the fluid transporting channels have a diameter in the range of 1 pm to 1000 pm. In another embodiment, the second gel material comprises Matrigel. In one embodiment, the permeable gel is a hydrogel. In another embodiment, the hydrogel is selected from the group consisting of agarose gel, gelatin, alginate, polypeptides, poly(acrylic acid), poly(vinyl alcohol), poly(ethylene oxide), and poly(ethylene glycol).

[0015] In one embodiment, the fluid transporting channels are molded in the gel using a mold made from a material selected from the group consisting of metal pins, plastic pins, metal wires, plastic wires, photosensitive materials, photoresists, 3D printable resins, and patterned sacrificial materials. In another embodiment, the fluid transporting channels have a shape that is selected from the group consisting of straight, curved, helical and branched. In one embodiment, the suction device is located at the top of the device. In another embodiment, the gel surrounds multiple defined pockets of a second gel material. Each pocket of second gel material comprises at least one volume for housing and culturing one or more organoids.

[0016] In another aspect of the present invention, a three-dimensional microfluidic device is provided. The device enables in vitro emulation of a blood brain barrier environment of a human body. The device includes an open-cell porous material surrounding a gel material. The gel material includes at least one volume for housing and culturing one or more organoids. The porousmaterial allows both direct circulation flow and diffusion flow into the gel material. Also, the open-cell porous material allows fluid flow at the size scale of whole organoids. The device also includes one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the open-cell porous material and in the gel. One or more types of organoids are cultured in the device. Further, the device is capable of emulating a microscopic and macroscopic blood flow pattern in the body and enables the study of how changes in the blood flow pattern affect the development of organoids.

[0017] In one embodiment, the device further includes a substrate with one or more embedded channels. Each embedded channel is in fluid communication with the open-cell porous material. The device further includes a suction device to remove fluid from the device. In another embodiment, the open-cell porous material is selected from the group consisting of polyurethane, polyamide, polyester, polypropylene, polyethylene, rubber, ceramics, and metal alloy. In one embodiment, the open-cell porous material surrounds multiple defined pockets of a gel material. Each pocket of gel material includes at least one volume for housing and culturing one or more organoids.

[0018] In another aspect of the present invention, a three-dimensional microfluidic device is provided. The device includes an array of fluid transporting channels formed in a permeable gel. The channels allow both direct circulation flow and diffusion flow from a channel into the gel. The diffusion flow is lateral to the direction of the channel and the channels are at the size scale of whole organoids. The device also includes one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the permeable gel. The device further includes one or more types of organoids cultured in the device. The device is capable of emulating a microscopic and macroscopic blood flow pattern in the body.

[0019] In another aspect of the present invention, a three-dimensional microfluidic device is provided. The device includes an open-cell porous material surrounding a gel material that includes at least one volume for housing and culturing one or more organoids. The porous material allows both direct circulation flow and diffusion flow into the gel material. The open-cell porous material allows fluid flow at the size scale of whole organoids. The device also includes one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the open-cell porous material and in the gel. Further, one or more types of organoids are cultured in the device. Also, the device is capable of emulating a microscopic and macroscopic blood flow pattern in the body and enables the study of how changes in the blood flow pattern affect the development of organoids.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0021] FIG. 1 A is a schematic showing an exploded view of a device according to the present invention.

[0022] FIG. IB is a schematic showing an assembled view of a device according to the present invention.

[0023] FIG. 1C is a schematic showing cross-sectional view with the suction lid detached from the substrate.

[0024] FIG. 2 is a schematic showing a device connection and flow circulation diagram of the entire system.

[0025] FIG. 3 is a schematic showing the device site for organoid culturing and the effect of the flow. The arrows show the flow pattern caused by the combination of vertical flow and lateral diffusion flow from the channel.

[0026] FIGs 4A-4G are a series of schematics showing the process flow of device fabrication, assembly, and usage.

[0027] FIG. 5 is a series of photos of fabricated parts.

[0028] FIG. 6A is a photo of a cross-sectional view of a molded microfluidic channel cut along the length of the channel (300 pm x 5 mm height),

[0029] FIG. 6B is a photo of a view from the top opening of the microfluidic channel after a 10 second diffusion of red-colored water into the agar gel from vertical flow inside the microfluidic channel.

[0030] FIG. 7 is a graph showing the vertical flow rate with respect to the main flow rate at different valve conditions (n=3 for the error bars).

[0031] FIGs 8 A-8F are a series of images showing the generation and characterization of blood vessel organoids derived from human embryonic stem cells and later used for preliminary tests of the 3D microfluidic device. FIG. 8 A shows mesoderm induction at day 5. FIG. 8B shows vascular lineage induction at day 10. FIG. 8C shows blood vessel organoid formation observed after day 15. FIG. 8D shows blood vessel organoid marked with GFP expressed in all cells. FIG. 8E shows CD31 expressed only in endothelial cells, and FIG. 8F shows DAPI taken from the fluid.

[0032] FIGs 9A and 9B are a pair of images showing preliminary blood vessel organoid culturing results in the 3D microfluidic device. FIG. 9A shows GFP, a protein that composes the blood vessel cells. FIG. 9B shows DAPI, a marker that the cells have taken from the fluid that circulated in the vertical channels and diffused to the organoid.

[0033] FIG. 10A is a schematic of alternative channel orientations.

[0034] FIG. 1 OB is a schematic of alternative channel geometries.

[0035] FIG. 11 is a schematic showing a vertical flow pattern generated using an example device made with porous materials.

[0036] FIG. 12 is a schematic showing a 3D model of an example device made with porous materials.

[0037] FIGs 13A-13D are schematics showing examples of circulation system configurations for batch culturing applications using multi chip implementation. FIG. 13 A shows devices connected in series. FIG. 13B shows devices connected in parallel. FIG. 13C shows devices connected both in series and in parallel. FIG. 13D shows a separate set of circulation components (i.e. pumps and tubing) used for each device to allow individual control of flow characteristics.

[0038] FIG. 14 is a schematic showing an embodiment of the present invention with four cavities in a single device chamber.

[0039] FIG. 15A-15C is a series of schematics showing examples of circulation system configurations for batch mode applications using single chip implementation (top view). FIG. 15A shows the same flow rate supplied to each culturing site using uniform design of distribution channels. FIG. 15B shows a different flow rate supplied to culturing sites by using separate sets of main flow inlets and outlets. FIG. 15C shows a different flow rate supplied to culturing sites by using branches from a single main flow channel and one set of main flow inlet and outlet. These branches may have different feature sizes or geometries to allow different flow characteristics (width difference illustrated).

[0040] FIG. 16A is a schematic showing an embodiment of the present invention with same sizes for the opening between the main flow channel and device chamber for each culturing site.

[0041] FIG. 16B is a schematic showing an embodiment of the present invention with different sizes for the openings between the main flow channel and device chamber for each culturing site.DETAILED DESCRIPTION OF THE INVENTION

[0042] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system -related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0043] The present invention involves a novel design of a 3D microfluidic device to emulate the environment to facilitate in vitro reconstruction of human blood brain barriers. Vertical circulation flows and lateral diffusion flows covering the entire size of the organoid are enabled by the device with adjustable flow rates. Preliminary culturing tests of blood vessel organoids showed that the organoid was properly activated in the device, proving that the environment was suitable for in vitro culturing with effective flow generation in the culturing site. Future experiments with the device will include long term co-culturing of brain organoids and blood vessel organoids.

[0044] The present invention involves a 3D microfluidic device to enable in vitro emulation of the flow environment where BBB is formed inside the body. An array of vertical channels is formed in high-permeability gel that allows both vertical circulation flow and lateral diffusion flow at the size scale of the whole organoids (FIG. 1 A). FIGs 1 A-1C illustrate the concept of the device 100. A substrate 150 with an embedded channel 310 replicates the artery, with a main flow going through the channel 310 horizontally. Portions of this main flow are diverged upward through a group of vertical microfluidic channels 320 formed in agar gel 130 that surrounds a center volume for housing and culturing the organoids 120 in Matrigel. These microfluidic channels 320 emulate the capillary blood vessels, and the high permeability of the gels allows efficient diffusion of the fluid from the vertical microfluidic channels 320 toward the organoids 120 for flow emulation and nutrition delivery, encouraging the development of BBB (FIG. 3). The fluid that reaches the top of the vertical channels into the suction lid 110 is drawn away from the suction lid 110 through the suction channel 300, emulating the vein. Referring to FIG. IB, in this embodiment of the present invention, the main flow inlet 200 and main flow outlet 210 of the substrate are offset by 90° from the air intake 220 and suction out-take 230 of the suction lid 110 for convenience of tubing connection. Two metal diaphragms 140 with through holes matched to the vertical channel locations are used to support the gels, with one attached to the substrate 150 and the other to the suction lid 110.

[0045] The component connection and flow circulation through the device 400 are illustrated in FIG. 2. The center of the device has Matrigel 450 containing agar gel 440. These gels are located between two diaphragms 470. Organoids 490 are located inside the agar gel 440. A peristaltic pump 420 drives the main flow through the main flow channel in the substrate 480. As the main flow reaches the bottom openings of vertical channels, hydraulic pressure diverges part of the flow upward through the entire length of the vertical channels. The fluid accumulated in the suction lid 460 is removed by negative pressure created by a suction pump 410. By adjusting the valve 430 at the air inlet to the suction lid, the flow rate through the vertical channels can be adjusted. Allculturing fluids are supplied from and returned to a single bottle reservoir with a special 4-port cap to minimize the setup footprint.

[0046] Regarding FIG. 3, a schematic of the artificial blood brain barrier 500 is shown. Vertical channels 510, allow fluid to pass through agar gel 540 in a vertical flow direction 550. As they pass through the vertical channel 510, molecules in the fluid 580 will diffuse into the agar gel 540. The diffused molecules 580 will move through the matrigel 530 and into the blood vessel cells 570 of the organoid 520. A BBB 560 is formed between the blood vessel cells 570 and the neuron 555.Fabrication Process and Results

[0047] The fabrication process of the device 600 is illustrated in FIGs 4A-4G. The parts, including the substrate 640, molding lid 610 and suction lid 740, were 3D printed using Formlabs Form 3B printer from the Formlabs BioMed resin, which is designated for biomedical devices with high requirement on biocompatibility (FIGs 4A-4G).

[0048] The orientations of the parts during the printing process were carefully chosen so that the hollow structures, such as the embedded channels, could be formed without supports typically required during this stereolithography 3D printing process. The molding lid 610 was used during fabrication to mold the agar gel 690 and form vertical channels 700 as well as the center open volume that was later filled with organoids 730 and Matrigel 720 for organoid culturing. The molding lid 610 and the substrate 640 include gel injection holes 620. Further, the substrate 640 has a main flow channel 630. Three metal diaphragms 650 were made from stainless steel using micro electro-discharge machining (pEDM). The metal is known to have good biocompatibility as well as high robustness to sustain the flow and molding process even at a thin 50 pm thickness. The diaphragms were patterned with 300 pm holes that match the diameter of the vertical microfluidic channels 700. The precision in size and location of these holes, as enabled by the use of the high precision pEDM, is important to the molding process to provide a tight fit between the holes and the pins used for channel molding, preventing gel leaking when the holes are too large or pin being blocked when the holes are too small. The pEDM provides a machining tolerance of <2 pm with high repeatability among micromachined diaphragms, which is important to the process. The size of the holes can be reduced further to 10s pm range to accommodate different needs of vertical flows.

[0049] After fabricating the 3D printed parts and the metal diaphragm, each of the 3D printed parts was assembled with a metal diaphragm 650 (FIG. 4B). For the molding lid 610, a cylindrical placeholder 660 was attached to the center of the diaphragm. This was used to form the center open volume that was later filled with organoids 730 and Matrigel 720. The need of the two geltypes comes from the curing requirement of agar gel. At the 2% concentration needed for its structural integrity, the agar gel has a curing temperature above 50°C, which is too high for the survival of organoids to be planted. Matrigel 720 can cure at room temperature and therefore was used for planting organoid 730 into the center open volume formed in agar gel 690 by the molding lid 610.

[0050] After assembling the molding lid 610 and the substrate 640, the holes on the two diaphragms were aligned, and a group of high-aspect-ratio pins 680 assembled on a pin holder 670 were inserted through the holes of both diaphragms (FIG. 4C). The positions on the pin holder 670 were also defined using pEDM to provide the necessary alignment accuracy.

[0051] The molding lid 610 was then rotated by a small angle to expose an injection hole 620 through the vertical sidewall of the cavity 710 on the substrate 640. A 2% agar gel solution heated to boiling was injected with a syringe (FIG. 4D). The syringe was pre-heated to prevent premature cool-down and cure of the agar gel. After the chamber was filled, the molding lid 610 was rotated back to the initial angle. After the agar gel was cured, the pin holder 670 along with the pins 680 as well as the molding lid 610 were removed (FIG. 4E). The organoid 730 was then planted into the center open volume along with Matrigel 720 to fill the volume (FIG. 4F). After the Matrigel 720 was cured, the suction lid 740 was attached to the top and the device was ready for fluid connections and culturing experiments (FIG. 4G).

[0052] FIG. 5 shows the 3D printed parts as well as the metal diaphragms micromachined by pEDM. The size of the substrate is 6^6 cm2. The cavity on the substrate has a diameter of 2.4 cm and height of 5 mm. Diaphragms with a circular array of 300 pm -diameter holes were attached to the center of the parts and were made from 50 pm thick stainless steel 304 using pEDM.Experimental Results

[0053] FIG. 6A shows the cross-sectional view of one of the vertical microfluidic channels molded in agar gel, cut along the length of the channel (300 pm x 5 mm height). Red colored water was circulated through the channels for 10 seconds to visualize diffusion of the fluid from the vertical channels. The resulting diffusion pattern from one channel is shown in FIG. 6B. The formation of the diffusion pattern over such a short time period indicates the high permeability of agar gel as expected, allowing the fluid to efficiently diffuse through the gel and reach the organoids. A COMSOL Multiphysics® simulation showed diffusion of pigment from flow inside vertical channel into surrounding gel, indicating good match with the experimental result shown in (b). “Transport of diluted species” model with porous medium (agar gel) used for simulation.

[0054] Characterization of the vertical flow rate under different main flow rates and valve conditions was performed with the assembled device. The valve at the air intake was set to threestates: fully closed, half open, and fully open. The air intake was at the maximum level when the valve was fully open, allowing minimum amount of negative pressure inside the suction lid for fluid removal. The main flow rate was 10 to 20 mL / min with an increment of 2.5 mL / min, approximately matching the range of the blood flow rate in human arteries. The vertical flow rate was measured by collecting the fluid that went through the tubing between the suction lid and the bottle reservoir for a given amount of time. The main flow volume that passed through the substrate was also collected to sum up with the vertical flow volume and compare with the supplied main flow volume from the peristaltic pump for cross-checking the accuracy of fluid collection. The vertical flow rate varying with the main flow rate and valve conditions is plotted in FIG. 7. The suction pump was set at the minimum suction power for all cases during the characterization. The results indicate that the vertical flow rate is under combined effect from both the hydraulic pressure of the main flow rate and the negative air pressure at the exit of the vertical channels. The vertical flow rate is proportional to the main flow rate and increases with decreasing air intake.

[0055] For preliminary organoid culturing test, human blood vessel organoids were generated from human embryonic stem cells (H9) by an optimized protocol based on previous practice. H9 line was labeled by green fluorescence protein (GFP) for better visualization of blood vessel cells and vessel network. FIG. 8A-8C shows a developmental timeline for blood vessel organoids. The mesoderm induction was finished in the first 5 days, followed by the vascular lineage induction from day 5 to 10, and blood vessel organoid formation was observed after day 15 (n = 3 culture). In addition to the GFP marker that exists in all blood vessel cells (FIG. 8D), red CD31 marker, which is only expressed in endothelial cells, was used to show the whole-mount staining of blood vessel organoids with endothelial cells derived from H9-GFP lines (FIG. 8E). A blue fluorescence dye, 4',6-diamidino-2-phenylindole (DAPI), was added to the static culturing fluid to mark the material interaction from the fluid into the organoids (FIG. 8F).

[0056] In preliminary organoid culturing tests to evaluate the device functionality and efficacy of vertical circulation and lateral diffusion of culturing fluid, a 30-day-old human blood vessel organoid, which had similar characteristics with the 15-day-old ones but further matured to a size of ~4 mm, was planted into the device center open volume and embedded with Matrigel (FIG. 9A). DAPI was added to the culturing fluid that was circulated from the bottle reservoir. The entire system except the two pumps was put into an incubator with 95% humidity at 37°C for an experiment lasting 1.5 hours. FIG. 9B shows the DAPI on the section of the organoid after the 1.5- hour perfusion, suggesting the entire organoid was soaked with the culturing fluid, proving the existence of a functional diffusion flow towards the blood vessel organoid form the vertical channels. This validated the primary function and capability of the 3D microfluidic device torealize flows at the size scale of organoids for emulation of the environment to facilitate BBB formation.Additional Applications of the Apparatus of the Present Invention

[0057] Besides the applications of in vitro BBB reconstruction, the 3D microfluidic devices of the present invention have a wide range of additional applications that contribute to various fields of research and technology. Notable examples include:

[0058] Drug Discovery and Development: 3D microfluidic devices provide a platform for studying the effects of drugs on human cells and tissues in a more accurate and realistic manner than traditional 2D cell culture. These devices can be used to test drug efficacy, toxicity, and pharmacokinetics, helping researchers identify potential candidates for further development.

[0059] Organ-on-a-Chip Systems: 3D microfluidic devices pave the way for creating organ- on-a-chip systems, which aim to mimic the structure and function of human organs. These systems enable researchers to study the interactions between different cell types and gain insights into organ-level physiology, disease mechanisms, and drug responses without the need for animal models.

[0060] Disease Modeling: With 3D microfluidic devices, researchers can create complex, multicellular models of various diseases, including cancer, neurodegenerative disorders, and infectious diseases. These models allow for the investigation of disease progression, cellular interactions, and response to therapeutics, aiding in the development of targeted treatments.

[0061] Tissue Engineering: 3D microfluidic devices provide a platform for engineering complex tissues by recreating the physiological microenvironment. They allow for precise control of cell positioning, nutrient delivery, and mechanical forces, enabling the development of functional tissues for transplantation, regenerative medicine, and studying tissue development and physiology.

[0062] Personalized Medicine: The use of 3D microfluidic devices in combination with patient-derived cells offers the potential for personalized medicine approaches. These devices can be used to test patient-specific drug responses, optimize treatment strategies, and develop personalized therapies based on an individual's unique genetic and physiological characteristics.

[0063] These are just a few examples of the diverse applications of 3D microfluidic devices beyond in vitro BBB reconstruction. As the technology continues to advance, it holds great promise for revolutionizing various aspects of research, healthcare, and industrial processes.Alternative Channel Configurations in Implementation with Hydrogel

[0064] In addition to the embodiment of the invention that demonstrated an array of 12 vertical channels molded with metal pins in agarose gel, additional channel array configurations can be used for the implementation of the device, including the use of different quantities, geometries, and / or orientations for the channels.

[0065] The orientations of channels can include slanted and horizontal channels besides vertical ones, as illustrated in FIG. 10A. Each of the channels can also be a combination of one or more segments in different orientations.

[0066] The geometries of the channels can include straight, curved, helical, branched, etc., as illustrated in FIG. 10B.

[0067] A variety of hydrogels can be used besides the agarose gel demonstrated. Other useful hydrogels include gelatin, alginate, polypeptides, poly(acrylic acid), poly(vinyl alcohol), poly(ethylene oxide), and poly(ethylene glycol).

[0068] To form the channels in gels through the molding method, the mold can be made from metal or plastic pins, wires of any material(s), photosensitive materials such as photoresists, 3D printable resins, patterned sacrificial materials, etc.Alternative Methods for Generation of Vertical Flow Patterns

[0069] Besides the method of generating vertical flows through channels formed in permeable hydrogels, vertical flow patterns can also be generated by applying pressure differences across open-cell porous materials. These materials can be polymer foams like polyurethane, polyamide, polyester, polypropylene, polyethylene, etc., as well as other porous materials such as rubber, ceramics, metal alloy, etc.

[0070] FIG. 11 shows a conceptual diagram of such an implementation method for a device 800. The porous material 810 is shaped to form a cavity that can be filled with cells or organoids 850 along with culturing fluid or gel 820. Device side walls 840 retain the porous material 810. The large quantity of open-cell pores in the porous material 810 will allow the formation of microscopic flow patterns traveling upwards when pressure differences are applied between the main flow channel 860 at the bottom and the exit channel 830 at the top. The flow characteristics are also affected by the porosity of the porous material 810, which is further determined by the mean size and density of the pores. By controlling these parameters, desirable flow patterns and flow rates can be generated and controlled. FIG. 12, where like numerals refer to like features, shows a 3D illustration of a potential implementation of the device built with this concept.System Configurations for Batch Culturing Applications

[0071] In some culturing applications, it is desirable to concurrently culture multiple groups of cells or organoids, either under the same conditions for statistical purposes, or under different conditions for comparison. Example applications may include drug discovery and development, disease modeling, personalized medicine, environmental monitoring etc. System configurations that allow such batch mode culturing applications can significantly increase the efficiency of the process and enable some types of experiments that cannot be realized otherwise. There are two ways to implement such batch configurations, either using multiple microfluidic chips or using a single chip with multiple culturing sites.Multichip Implementation

[0072] In the multichip implementation, a group of multiple microfluidic devices, each with one or more culturing sites, are assembled into an array to perform the batch mode culturing process. As illustrated in FIGs 13 A-13D, the circulation components, i.e. the peristaltic and suction pumps, for each microfluidic device can be either one single set or multiple sets. The microfluidic devices can be connected in series, parallel or both. Some of these example configurations, such as those shown in FIG. 13A-13C, can only allow the same flow characteristics among the devices unless devices of different fluidic features are used, while others, as shown in FIG. 13D, can allow separate control of the flow characteristics in each device.Single-chip Implementation

[0073] In the single-chip implementation, multiple culturing sites can be integrated onto a single device substrate and the circulation is designed such that either the same flow rate is supplied to each site (e.g., the configuration in FIG. 15 A), or different flow rates can be generated using separate (FIG. 15B) or joint main flow inlet / outlet (FIG. 15C). The device chambers in FIGs 15A- 15C can be either single-cavity chambers or multi-cavity chambers, or a combination of these two.

[0074] In another design shown in FIG. 14, a chip 1000 can contain a single device chamber 1030 on a substrate 1070. The device chamber 1030 includes multiple cavities 1010 for placement of multiple organoids 1020 in the chamber 1030. This embodiment shows four cavities in a single device chamber. The device chamber 1030 is connected to main flow channels 1050, with a main flow inlet 1060 and a main flow outlet 1040.

[0075] These cavities can be designed to experience either identical or different flow rates by using microfluidic features on the flow path, e.g. either the same or different sizes for the opening between the main flow channel and device chamber for each culturing site as shown in FIGs 16Aand 16B. Referring to FIG. 16A, a device 1100 has two cavities (culturing sites) 1110 containing organoids 1120. A device chamber side wall 1130 is shown. This device 1100 has the main flow 1150 feeding openings of same size 1140. Referring to FIG. 16B, device 1200 has two cavities (culturing sites) 1210 containing organoids 1220. A device chamber side wall 1230 is shown. This device 1200 has the main flow 1250 feeding openings of different sizes 1240.

[0076] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

WHAT IS CLAIMED IS:

1. A three-dimensional microfluidic device for in vitro emulation of a blood brain barrier environment of a human body comprising: a. an array of fluid transporting channels formed in a permeable gel, wherein the channels allow both direct circulation flow and diffusion flow from a channel into the gel, wherein the diffusion flow is lateral to the direction of the channel and the channels are at the size scale of whole organoids; b. one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the permeable gel; c. one or more types of organoids cultured in the device, wherein the device is capable of emulating a microscopic and macroscopic blood flow pattern in the body and enables the study of how changes in the blood flow pattern affect the development of organoids.

2. The device of claim 1 wherein the organoids comprise both brain organoids and blood vessel organoids and the brain organoids and blood vessel organoids are cocultured together.

3. The device of claim 1 wherein the one or more fluid movement devices are pumps.

4. The device of claim 1 comprising a substrate with one or more embedded channels, each embedded channel being in fluid communication with the fluid transporting channels, wherein the gel surrounds a second gel material that comprises at least one volume for housing and culturing one or more organoids, and further, wherein the device comprises a suction device to remove fluid from the device.

5. The device of claim 4 wherein the permeable gel and second gel material are supported on one or more diaphragms, and further, wherein the one or more diaphragms have a plurality of holes, the holes being matched to the locations of the fluid transporting channels.

6. The device of claim 5 wherein the fluid transporting channels have a diameter and further, wherein the holes in the one or more diaphragms have a diameter that is the same as the diameter of the fluid transporting channels.

7. The device of claim 6 wherein the holes in the one or more diaphragms have a diameter in the range of 1 pm to 1000 pm.

8. The device of claim 6 wherein the fluid transporting channels have a diameter in the range of 1 pm to 1000 pm.

9. The device of claim 1 wherein the second gel material comprises Matrigel.

10. The device of claim 1 wherein the permeable gel is a hydrogel.

11. The device of claim 10 wherein the hydrogel is selected from the group consisting of agarose gel, gelatin, alginate, polypeptides, poly(acrylic acid), poly(vinyl alcohol), poly(ethylene oxide), and poly(ethylene glycol).

12. The device of claim 1 wherein the fluid transporting channels are molded in the gel using a mold made from a material selected from the group consisting of metal pins, plastic pins, metal wires, plastic wires, photosensitive materials, photoresists, 3D printable resins, and patterned sacrificial materials.

13. The device of claim 1 wherein the fluid transporting channels have a shape that is selected from the group consisting of straight, curved, helical and branched.

14. The device of claim 4 wherein the suction device is located at the top of the device.

15. The device of claim 4 wherein the gel surrounds multiple defined pockets of a second gel material, wherein each pocket of second gel material comprises at least one volume for housing and culturing one or more organoids.

16. A three-dimensional microfluidic device for in vitro emulation of a blood brain barrier environment of a human body comprising: a. an open-cell porous material surrounding a gel material that comprises at least one volume for housing and culturing one or more organoids, wherein the porous material allows both direct circulation flow and diffusion flow into the gel material, wherein the open-cell porous material allows fluid flow at the size scale of whole organoids; b. one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the open-cell porous material and in the gel; wherein one or more types of organoids are cultured in the device, and further, wherein the device is capable of emulating a microscopic and macroscopic blood flow pattern in the body and enables the study of how changes in the blood flow pattern affect the development of organoids.

17. The device of claim 16 comprising a substrate with one or more embedded channels, each embedded channel being in fluid communication with the open-cell porous material, wherein the device further comprises a suction device to remove fluid from the device.

18. The device of claim 16 wherein the open-cell porous material is selected from the group consisting of polyurethane, polyamide, polyester, polypropylene, polyethylene, rubber, ceramics, and metal alloy.

19. The device of claim 16 wherein the open-cell porous material surrounds multiple defined pockets of a gel material, wherein each pocket of gel material comprises at least one volume for housing and culturing one or more organoids.

20. A three-dimensional microfluidic device comprising: a. an array of fluid transporting channels formed in a permeable gel, wherein the channels allow both direct circulation flow and diffusion flow from a channel into the gel, wherein the diffusion flow is lateral to the direction of the channel and the channels are at the size scale of whole organoids; b. one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the permeable gel; c. one or more types of organoids cultured in the device, wherein the device is capable of emulating a microscopic and macroscopic blood flow pattern in the body.

21. A three-dimensional microfluidic device comprising: a. an open-cell porous material surrounding a gel material that comprises at least one volume for housing and culturing one or more organoids, wherein the porous material allows both direct circulation flow and diffusion flow into the gel material, wherein the open-cell porous material allows fluid flow at the size scale of whole organoids; b. one or more fluid movement devices capable of controlling the circulation flow to enable control of flow pattern in the open-cell porous material and in the gel; wherein one or more types of organoids are cultured in the device, and further, wherein the device is capable of emulating a microscopic and macroscopic blood flow pattern in the body and enables the study of how changes in the blood flow pattern affect the development of organoids.

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