Microphysiological system for neural modeling

Abstract

Systems and methods for neural modeling are disclosed. A perfusable microfluidic platform with human hippocampal organoid integration and functional vasculature is disclosed. Vascularized human brain organoids can be developed on a microphysiological platform. The system can include a membrane configured to enable perfusion throughout the membrane. The membrane can couple with a carrier. The system can include the carrier including channels. The channel can be configured to deliver a first set of cells to the membrane through a lumen of the membrane; and deliver a second set of cells to the membrane through an exterior of the membrane. The system can include monitoring components to continuously and non-invasively measure the onset and development of neural oscillations.

Description

MICROPHYSIOLOGIC AL SYSTEM FOR NEURAL MODELINGCROSS-REFERENCE TO RELATED APPLICATIONS[00011 This application claims the benefit of U.S. provisional application Serial No. 63 / 655,234 filed June 3, 2024, the disclosure of which is hereby incorporated in its entirety by reference herein.TECHNICAL FIELD

[0002] Aspects of the disclosure generally relate to systems and methods for simulating and monitoring neural activities.BACKGROUND

[0003] Neural activity can be simulated and monitored. Observing brain activity to determine patterns in neural activity can prove difficult due to inherent challenges in accessing a functioning brain (e.g., surgery, complications thereof, etc.) or in monitoring an accurate model of a brain. Different models for simulating behavior of a brain can include biological models, computer models, electronic models, or combinations thereof.SUMMARY

[0004] Presented herein are systems and methods for a microphy si ologi cal system to model neural activity. In some cases, the system can include multiple subsystems. The subsystems can include a microfluidics-based neural microphy si ologi cal system, an integrated arrays to monitor neural activity, and / or a blood-brain barrier modeling system.(0005] The system can mimic a blood-brain barrier through its structural geometry enabling ordered perfusion of cells and materials. Through the systems and methods disclosed herein, the generation of an enhanced model of brain organoids, such as the hippocampus, with vascularization is discussed.

[0006] Modeling organoids of the brain and the brain itself can pose several challenges. The movement of materials (e.g., ions, enzymes, molecules, other chemical compositions) and cells, among others, is regulated in part by the blood-brain barrier. The blood-brain barrier is a semi- permeable membrane between the brain and blood vessels which regulates transport of materials, cells, etc. between the brain and the blood. Modeling vasculatures of the brain such as the bloodbrain barrier (BBB) can pose challenges due to the complexities of assembling a synthetic membrane to be permeable and allow diffusion in a manner that accurately simulates brain function. These challenges can include maintaining mechanical stiffness and shear stress of the BBB, among others.

[0007] Accurately modeling the vasculature of the brain can help to identify neural development and maturation via molecular signaling, tight control of transported species across the BBB, and the transport of oxygen, nutrients and the removal of waste. Therefore, it is desirable to provide an accurate model of neural behavior which can mimic the complexities of the brain, such as the vasculature, various environments (e.g., in vivo, among others), neural networks, etc.|0008| To account for these and other technical challenges, the systems and methods described herein can simulate a brain or brain organoid including a blood-brain barrier. The systems and methods described herein can provide for including relevant BBB cell types to simulate a BBB through various channels and a membrane geometry to enable perfusion. For example, the systems and methods described herein can include a microfluidic MPS platform with a 3D bio-printed neurovasculature. The MPS platform can include a lid with micropumps and / or electrodes disposed therein to support long term culture and functional development of neural brain specific regions, as well as enable monitoring of the neural brain specific regions.

[0009] The microfluidic MPS platform can enable delivery of relevant BBB cell types, nutrients, enzymes, as well as other materials and cells. By including these relevant BBB cell types, such as astrocytes, pericytes, and endothelial cells, the system can lead to higher expression of tight junction proteins, lower permeability, and higher expression of tight junction proteins versus other conventional brain-modeling systems, such as endothelial cells alone in self-assembled vascular structures in fibrin matrices. For example, the systems and methods described herein can includea 3D bio-printed perfused neurovascular structure consisting of an extracellular matrix (ECM) such as hydrogel. In some cases, this structure can be disposed on a carrier with embedded microfluidic channels supporting perfusion.

[0010] The systems and methods disclosed herein can include a monitoring system. The monitoring system can be a non-contact electrophysiological monitoring system. Non-contact electrophysiological monitoring as disclosed herein can enable monitoring of neural functional development as a function of neural differentiation and model perturbations. The scalability of the MPS described herein can enable in situ sensing to monitor neural circuit functional development.

[0011] Some aspects of the technical solutions described herein can include a system for simulating a blood brain barrier. The system can include a membrane. The membrane can enable perfusion throughout the membrane. The membrane can couple with a carrier. The system can include the carrier. The carrier can include one or more channels. The one or more channels can deliver a first set of cells to the membrane through a lumen of the membrane. The one or more channels can deliver a second set of cells to the membrane through an exterior of the membrane.

[0012] In some aspects, the system can include a lid including micropumps. The micropumps can provide materials to the membrane. In some aspects, the membrane can be disposed on the carrier. In some aspects, the first set of cells can include one or more of astrocytes, pericytes, or endothelial cells. In some aspects, to deliver the first set of cells, the channels can provide the first set of cells to the membrane according to a sequence. In some aspects, the carrier can couple to a microphy si ologi cal platform. In some aspects, the one or more channels can define a first set of fluid flows to the membrane and a microphysiological platform can define a second, separate set of fluid flows to the membrane. In some aspects, the system can include a lid including micropumps. The micropumps can pressurize the first and second set of fluid flows. In some aspects, the micropumps can control one or more mechanical or biological stresses within the membrane. In some aspects, the system can maintain a neural organoid.

[0013] Some aspects of the technical solutions described herein can include a microphysiological system for simulating a blood brain barrier. The membrane can include a structure arranged to enable perfusion throughout the membrane. The system can include a carrier. The carrier caninclude one or more channels. The one or more channels can deliver a first set of cells to the membrane through a lumen of the membrane. The one or more channels can deliver a second set of cells to the membrane through an exterior of the membrane. The system can include one or more chambers configured to couple to the carrier to sustain one or more neural organoids.[0014| Some aspects of the technical solutions described herein can include a monitoring system for a neural microphy si ologi cal platform. The system can include electrodes configured to couple with a lid including micropumps. The electrodes can enable continuous monitoring of a neural organoid.BRIEF DESCRIPTION OF THE DRAWINGS|0015| The foregoing and other objects, aspects, features, and advantages of the disclosure will become more apparent and better understood by referring to the following description taken in conjunction with the accompanying drawings. The accompanying drawings are not intended to be drawn to scale. Like reference numbers and designations in the various drawings indicate like elements. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0016] FIG. 1 illustrates a block diagram of an example neural modeling system;

[0017] FIG. 2 illustrates a perspective view of the example neural modeling system.

[0018] FIG. 3 illustrates a perspective view of one neural model of FIG. 2.

[0019] FIG. 4 illustrates a perspective view of the carrier of FIG. 3.

[0020] FIGs. 5A-C illustrate the carrier 110 in sequential fabrication order.

[0021] FIG. 6A illustrates a cut-away view with exploded portions of the model system.

[0022] FIG. 6B illustrates a vascular model in an MPS showing high shear can cause tissue alignment.

[0023] FIG. 6C illustrates vascular designs following Murray’s law to balance flow.

[0024] FIG. 6D illustrates a model of a perfusable carrier and bifurcating vascular feasibility design.

[0025] FIG. 6E illustrates an image of a 3D bioprinted bifurcating vascular design on a bioprinted perfusable carrier.

[0026] FIG. 6F depicts a fluorescence image of perfused 3 pm fluorescent beads in bioprinted vasculature.

[0027] FIG. 7 illustrates an example chart illustrating effects of shear stress forces on a brain microvascular endothelial cells barrier function as measured by TEER (Transepithelial / Transendothelial Electrical Resistance).

[0028] FIG. 8A illustrates the toxicokinetic impact of MPP+ on midbrain dopaminergic signaling. FIG. 8B illustrates distinct EEG (Electroencephalography) and LFP (Local Field Potential) patterns and corresponding brainwaves before and during chronic MPP+ application.

[0029] FIG. 8C illustrates time-averaged brainwave power trends of midbrain organoids during MPP+ exposure.|0030] FIG. 8D illustrates phase-amplitude coupling (PAC) analysis for brainwave alterations associated with MPP+ exposure, indicating abnormal delta-beta coupling during the MPP+- induced hyperactivity.

[0031] FIG. 8E depicts a temporal trace of sorted MI values during the MPP+ exposure.

[0032] FIG. 9 illustrates an example of IHC (Immunohistochemistry) images of a hippocampus organoid.DETAILED DESCRIPTION

[0033] Reference will now be made to the embodiments illustrated in the drawings, and specific language will be used here to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Alterations and further modifications of the features illustrated here, and additional applications of the principles as illustrated here, which would occur to a person skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the disclosure.

[0034] Described herein are systems and methods for neural modeling. Through the systems and methods disclosed herein, the generation of an enhanced model of brain organoids with vascularization is provided. The systems and methods disclosed herein can include non-contact monitoring of brain development and function.

[0035] The systems and methods disclosed herein can integrate biological, physical, and material cues of a brain or brain organoid with in situ monitoring of neural circuits in a scalable MPS platform. The described systems and methods can provide a BBB (blood-brain-barrier) with the required cell types and architecture. The BBB is a highly selective barrier formed by endothelial cells lining brain capillaries. More generally, human brain organoids are three-dimensional tissues generated in vitro from pluripotent stem wells that recapitulate the early development of the human brain. Brain organoids consist mainly of neural lineage cells such as neural stem / precursor cells, neurons, astrocytes, and oligodendrocytes. These organoids, however, lack vasculature, which plays a role in brain homeostasis and development. The vascular system and the BBB may regulate neural differentiation, migration and circuit formation during development, regulate nutrient and waste transport, maintain homeostasis, etc. Vascularization may initiate approximately 30 days after fertilization, and defects thereof may result in brain malformations. Human organoid modeling may provide researchers with a better understanding of the impact of the BBB on hippocampal organoid development and function.[0036| The created BBB can be disposed on a 3D printed vascular lumen to control the mechanical stiffness and shear stress important for BBB function. Through the systems and methods disclosed herein, the development and maturation of a brain-specified region can besupported. Perfusion and BBB-neural integration in a 3D architecture may be able to increase neural cell differentiation and proliferation rates, decrease hypoxia and necrosis, enable heterotypic (i.e. endothelial- epithelial) cellular interactions and crosstalk leading to a more physiologic tissue architecture and function than conventional brain-modeling systems.

[0037] Multiple subsystems contribute to the development of a microphysiological system (MPS) for neural modeling. These subsystems can include a first system directed to a microfluidics-based neural MPS with synthetic vasculature integration, a second system directed to integrated arrays for monitoring neural activity, and a third system directed to blood-brain barrier modeling. Each of these systems is described herein under the following subheadings:|0038| Section A: Microfluidics-Based Neural Microphysiological System

[0039] Section B: Integrated Arrays to Monitor Neural Activity

[0040] Section C: Blood-Brain Barrier Modeling

[0041] Each of these subsystems can include any of the components of the others, as well as components not detailed herein. Further, the MPS system for neural modeling can include more, fewer, or different subsystems than those described herein. The various subsystems can interact, communicate, or otherwise be coupled with one another to provide the MPS for neural modeling.Section A: Microfluidics-Based Neural MPS[0042) FIG. 1 illustrates a block diagram of an example neural modeling system 100. The neural modeling system 100 can include a culture plate 105, a membrane 115, a carrier 110, and a lid 120 including micropumps 125. In brief overview of the system 100, the culture plate 105 can interface with the membrane 115 and the carrier 110 to provide various cells and materials to a tissue contained within the MPS. In some cases, the lid 120, including the micropumps 125, facilitates the transport of these materials and cells.

[0043] FIG. 2 illustrates a perspective view of the example neural modeling system 100 having a plurality of neural model 103. Referring to FIGs. 1 and 2, the culture plate 105 may include a well plate 130 configured to accept the carrier 110 (not labeled in FIG. 2) and / or the lid 120.

[0044] FIG. 3 illustrates a perspective view of one neural model 103 of FIG. 2. In some cases, the neural model 103 can include one or more chambers, or reservoir wells 140, which can hold one or more tissues. The tissue can be one or more types of cells, such as brain cells, liver cells, blood cells, among others. The wells 140 could also hold tissue medias and contain circulating immune cells or Endothelial cells on an inner lumen. In some cases, the reservoir wells 140 (also referred to as “media wells”) are fluidly connect with the carrier 110 to provide one or more fluid flows to the membrane or to the tissue enclosed within the culture plate 105.

[0045] The culture plate 105 can be included in or coupled to a microphy siological system (MPS), such as the neural modeling system 100. The miniaturized in vitro model may mimic the function of human neural tissues. As explained, the system may aim to mimic aspects of the human central nervous system, and the BBB.

[0046] In some cases, as illustrated in FIG. 3, the MPS includes reservoir wells 140. This can enable two or more sets of independent fluid flows. For example, this coupling can allow a first set 140a of two media wells to connect to a fluidic circuit of the carrier 110 and leave a second set 140b of two media wells available for an additional mixing flow external to the tissue or as a second perfusion circuit for the membrane 115 to develop gradients of materials such as morphogens or analytes in the tissue. One of each of the pairs of the wells 140 may be arranged on opposite sides of the carrier 110. Although pairs are described herein, a single or other number of wells, including an odd number, could be contemplated. Such descriptions of the pairs are for example purposes.

[0047] The reservoir wells 140 can contain different media types as required. The micropumps 125 (also referred to herein as the microfluidic pumps 125) are described herein and are configured to pass the fluid from one media reservoir 140 to a matching paired media reservoir 140 creating a pressure head to drive a recirculating flow. The micropumps 125 are designed to balance fluid shear stress, pressure, and flow rates, to enable endothelial alignment, tight junction formation and cellular remodeling while also establishing appropriate nutrient, oxygen, and morphogen delivery within the tissue.

[0048] FIG. 4 illustrates a perspective view of the carrier 110 of FIG. 3. FIGs. 5A-C illustrate the carrier 110 in sequential fabrication order. The carrier 110 can be a structure configurable to attach to the membrane 115 (illustrated in FIG. 4 and 5C) and including one or more channels 150. In some cases, the carrier 110 is a rigid structure, while in other cases the carrier 110 can expand or contract to accommodate growth of the tissue. In some cases, the carrier 110 is a translucent or semi-translucent material such as plastic or glass. The carrier 110 can couple with the culture plate 105. In some cases, this coupling can enable microfluidic perfusion of the membrane 115 to control BBB shear stress and the transport of critical growth factors, nutrients, oxygen, and waste via the lid 120. By controlling shear stress of the BBB and transport of materials, hypoxia can be reduced and neural differentiation and growth can be increased.[0(149] In some cases, the carrier 110 can be coupled to the membrane 115. As explained, the carrier 110 can include one or more channels 150, microfluidics, or other structures which enable a flow of two or more fluids containing materials and / or cells to the membrane 1 15 and / or the tissue.|0050| The membrane 115 can be a synthetic perfusable tissue, such as a vascular structure. In some cases, the membrane 115 perfuses fluids similarly to or in a manner mimicking an organ or organoid, such as a blood-brain barrier or lumen. The membrane 115 can include or be formed of an extracellular matrix like materials such as hydrogel. In some cases, the membrane 115 can cause capture of tissues to enable growth of the tissue. A retaining ring may be configured to hold tissues, cells, or other materials within the membrane 115.

[0051] Coupling the carrier 110 and the membrane 115 can generate a perfused neural organoid (such as a hippocampus) with the BBB by sequentially seeding various neural cell-types such as astrocytes, pericytes, and endothelial cells in a lumen of the membrane 115 followed by seeding an exterior of the coupled carrier 110 and membrane 115 structure with deposited iPSC cells within the retaining ring or other retaining region of the membrane 115.100521 Referring specifically to FIGs. 5A-C, a sequential workflow of fabrication of a coupled structure including the membrane 115 and the carrier 110 is illustrated. In some cases, the carrier 110 and / or the membrane 115 can be constructed by additive manufacturing techniques such as3D printing. In some cases, the additive manufacturing can include bio-inks. This can enable the formation of the channels 150 (also referred to herein as perfusion channels) or other structures in the carrier 110 and / or the membrane 115 for perfusion. In some cases, the carrier 110 is printed separately from the membrane 115 and attached to the membrane 115. In some cases, the membrane 115 is printed in conjunction with the carrier 110. In the example fabrication show, the carrier 110 may be printed via 3D printing or other techniques, as illustrated in FIG. 5 A. In FIG. 5B, the membrane may include a vasculature and / or iPSC (induced pluripotent stem cell) retaining ring that may be arranged at an opening 170 defined by the carrier 110. In FIG. 5C, an organoid 165 is arranged within the opening at the vasculature 160.

[0053] The channels 150 can establish gradients of materials such as biological factors, cells, among others. In some cases, by including multiple perfusion channels to establish gradients of biological factors, patterning developing organoid tissue can be improved compared to some conventional systems in which organoids of different regions are physically coupled together after differentiation. For example, the channels 150 can enable development of an axis of differentiation, multiple brain regions differentiated together in one organoid, among others, by delivering different spatially defined factors. In one example, the channels 150 include at least one or two channels 150 to generate a first fluid flow to the membrane and a second, separate fluid flow to the membrane

[0054] In some cases, the bio-ink includes physiologically relevant mechanical stiffness, morphogen, and nutrient transport properties, enabling cellular interactions and remodeling. In some cases, the printed geometry (e.g., structure) of the membrane 115 can be printed to enable a vascular geometry and flow rates to mimic an organoid or organoid by creating physiologic luminal shear stress and allowing spatial and temporal delivery of distinct morphogens and nutrients. In some cases, the 3D printing is two-photon printing which enables micron scale voxel resolution for the physiologically relevant arteriole / venule length scales of 10-90 pm.[0055| The bio-ink can include one or more cells. For example, cells can be included in the bioink and thereby disposed through the membrane 115 during printing of the membrane 115 with the bio-ink including the cells. In some cases, one or more cells can be placed, seeded, or otherwisesustained on the membrane 115, the carrier 110, or a combination thereof. In some cases, a first set of cells can be included in the bio-ink and arranged within the membrane 115 or the carrier 110 and a second set of cells can be seeded on the membrane 115 or the carrier 110.

[0056] In some cases, it can be desirable to allow cells or other materials to perfuse through the structures of the membrane 115. The membrane 115 can include a lumen. The lumen can be a perfusion structure within the membrane. For example, endothelial cells can be seeded into the lumen under perfusion (ramp to 10 dyn / cmA2 over days, for example) to examine the cellular morphology and confluency. For example, iPSC cells can be perfused through the bio-ink structures (e.g., the carrier 110 and / or the membrane 115).

[0057] In some cases, the bio-ink can support a BBB and / or neural tissue culture. The bio-ink can include properties such as physiologically relevant mechanical stiffness, morphogen and nutrient transport properties, cell adhesion properties, and ability to be re-modeled by cells, among others. In some cases growth factors can be delivered to cause cells to re-model printed vasculature to sprout new vessels. Further, the bio-ink can include GELMA, HAMA or a mixture thereof. In some cases, the bio-inks are biodegradable. In some cases, the bio-inks have a stiffness range of range l-100kPa.

[0058] In some cases, the structure of the carrier 110 and / or membrane 115 can be affected by properties of the bio-inks and printing method such as % weightwolume, ratios, laser power & layer height, scan speed, degree of methacrylation (DOM), and photoinitiator concentration, such as LAP . In addition, post-printing enzymatic treatments (Haulyronidase (Hase)) of bioprinted structures may be employed to circumvent the usual trade-off between the need for low material stiffness for cellular viability and physiologic functionality and higher stiffnesses required for printability. In some cases, the bio inks can include porcine Type A GelMA, 50%, 95% DOM, 300 bloom, HAMA 50 or 95% DOM, among others. In some cases, the printed carrier 110 and / or membrane 115 exhibit mechanical compression properties targeting a soft tissue stiffnesses of order 10 kPa.

[0059] Referring again to FIGs. 1-4, the carrier 110 and / or the membrane 115 can control the tissue biochemical and mechanical environment during development of a tissue. The structure ofeach of the carrier 110 and the membrane 115 can enable control over fluid mechanics, chemical transport, and solid mechanics. In some cases, the membrane 115 is within a threshold distance of the tissue. The threshold distance can be, for example, 100-300pm. In some cases, maintaining a threshold distance between the membrane 115 and the tissue can minimize hypoxia and establish appropriate nutrient, oxygen, and biological factor concentrations within the tissue as compared to conventional systems.

[0060] As shown in FIG. 7, the carrier 110 and / or the membrane 115 can balance fluid shear stress shown to cause endothelial alignment and enhance endothelial tight junction formation as compared to conventional systems. The membrane 115 and / or the carrier 110 can control any fluid flows with the lid 120 including the micropumps 125.[00611 In some cases, the lid 120 can cause a flow of fluid through the one or more channels 150 by the micropumps 125. The lid 120 can provide a closed environment around the tissue in conjunction with the culture plate 105. The lid 120 can include two or more independent pneumatically driven precision micropumps 125 (also referred to herein as the microfluidic pumps 125) which pass the fluid from one media reservoir 140 to a matching paired media reservoir 140 creating a pressure head to drive a recirculating flow.

[0062] The micropumps 125 can balance fluid shear stress, pressure, and flow rates, to enable endothelial alignment, tight junction formation and cellular remodeling while also establishing appropriate nutrient, oxygen, and morphogen delivery within the tissue. In some cases, scaffold strains of less than 10% can remain in the elastic region of the membrane 115.|0063[ As a first order example to demonstrate feasibility of the design space, the maximum radial strain can be estimated for a thin cylindrical vessel as;

[0064] For this example, the values, L= vessel length (2 mm), T= shear stress (1 Pa), v= Poisson’s Ratio (0.4), E= Young’s Modulus (10 kPa), t=vessel thickness (20 pm), the max strain Emaxis well below 10% strain with an 8max 8e-3.[0065| As explained, FIGS 2-5 depict an MPS system configurable to accept a microfluidic culture plate 105 (MCP) and 3D synthetic Neurovascular (3D-sNv) to enable a perfusable BBB- neural organoid. The 3D-synthetic Neurovascular (3D-sNv) can be like or include the membrane 115. The membrane 115 can include 3D-sNv bio-ink materials with a structure (e.g., geometry) to enable control over a mechanical environment (luminal shear stress, mechanical stiffness, cellular adhesion, cellular remodeling) as well as a biochemical environment (media, morphogens, oxygen, waste removal) for a tissue. FIG.3 further depicts perfusable flows across a membrane (e.g., the membrane 115) enabled by a lid (e.g., the lid 120) and micropumps therein (e.g., the micropumps 125).

[0066] FIGS. 6A-F illustrate several views of a microfluidics-based neural MPS with an acellular vasculature including hydrogel printed into a lumen structure, or in another example, with synthetic vasculature integration (sNv-MPS) and related bioprinting. Such acellular vasculatures may be printed or molded to mimic a blood vessel and allow perfusion therethrough. sNv-MPS may be a combination of synthetic vasculars with tissue constructs and may mimic perfusable channels and barrio functions.

[0067] FIG. 6A illustrates a cut-away view with exploded portions of the model system 100.

[0068] FIG. 6B depicts a vascular model in an MPS showing high shear can cause tissue alignment.

[0069] FIG. 6C depicts vascular designs following Murray ’ s law to balance flow. In some cases, these vascular designs can be a geometry of the membrane 115. In some cases, the membrane 115 features a bifurcated vascular design.

[0070] FIGS. 6D-F depict an example 3D bioprinted carrier approach. In this example, using a 2 photon bioprinter, a carrier structure (e.g., the carrier 110) can be printed with the embedded microfluidic channels 150 with a bifurcating vascular structure (e.g., the membrane 115) insequential steps and shown to be functionally perfusable. FIG. 6D depicts a model of a perfusable carrier (e.g., the carrier 110) and bifurcating vascular feasibility design. FIG. 6E depicts an image of a 3D bioprinted bifurcating vascular design (e.g., the membrane 115) on a bioprinted perfusable carrier (e.g., the carrier 110). FIG. 6F depicts a fluorescence image of perfused 3 pm fluorescent beads in bioprinted vasculature (e.g., vasculature 160).

[0071] FIGs. 2-5 show an example conceptual model of a system for neural modeling This may be an example based on a 384 well plate where the well walls are not shown. FIG. 3 describes an example method of production and operation of the neural monitoring system. A subset of wells 140 (in this example, 8) of a well plate (such as of the culture plate 105) can include a single unit cell or BBB-hippocampal model of the system. FIG. 5 depicts a transparent carrier (e.g., the carrier 110) printed in, for example, UpOpto ink followed by printing the vasculature 160 (e.g., the membrane 115) in, for example GelMA / HAMA ink, and iPSC retaining structure (GelMA / HAMA ink). The carrier 110 can be attached to the culture plate 105 to form a perfusable fluidic seal. Cells, fluids, or cultures, among others (e.g., iPSC cells) can be deposited on top of the 3D-SNV structure (e.g., the membrane 115) within the retaining structure (e.g., of the membrane 115) to allow formation of the hippocampus organoid (or other organoids). The retaining structure can be printed with a low cross-linked Gelatin to allow dissolution over time. The lid (e.g., the lid 120) can include the pumps (e.g., the micropumps 125) for the perfusion of the tissue as well as the electrodes to monitor the brain electrophysiology.[00721 By the systems and methods described herein, a neural monitoring platform, such as an sNv-MPS platform with a workflow enabling a flexible snap-in 2 photon bio-printed 3D-sNv carrier design incorporating at least two independent perfusion circuits controlled by micropumps located in the lid enabling control of luminal shear stress and morphogen and growth factor gradients in neural tissues, is provided. The system can multiply tissue models (e.g., 24, 48, 100), thereby enabling robust statistical designs and investigations and be ready for integration of a BBB vasculature (e.g. vasculature 160) and neural iPSC tissues.Section B: Integrated Arrays to Monitor Neural Activity[00731 Monitoring organs or organoids such as the hippocampus functions can be desirable. Monitoring simulated organs or organoids can exhibit functionality of the simulation as an organ mimic. Monitoring simulated organs can enable statistical, experimental, and other analyses of the organ in response to various stimuli. The systems and methods described herein can integrate noncontact EEG arrays on a neural MPS to monitor brain rhythms noninvasively. Given that neural oscillations and synchrony are fundamental mechanisms for sensory and cognitive processes in health and disease, the systems and methods described herein include a low 1 / f noise bioelectronic system and integrated microfluidic MPS that allows real-time monitoring of brainwaves. In some cases, the integrated microfluidic MPS can be like or include the system and methods described in Section A. For example, the integrated microfluidic MPS can include the neural modeling system 100.

[0074] The integrated microfluidic MPS can include a lid 120 containing a non-contacting electrode array. This design can minimize direct cell interaction as compared to conventional systems, thus supporting unimpeded organ / organoid development while remotely monitoring brainwave activities. Through this integration, commencement of neural oscillations and how they interconnect and influence each other in hippocampal organoids can be monitored. In some cases, the integrated microfluidic MPS includes an integrated low 1 / f noise-based EEG bioelectronic array on the lid of the brain organoid MPS.

[0075] In some cases, the lid 120 of FIG. 1 can include one or more electrodes (also referred to herein as surface electrodes). The electrodes can be high-density surface electrodes for non-contact field potential readout. In some cases, the electrodes can enable monitoring, sensing, or other readouts of a tissue, such as a tissue grown within the system of Section A. In some cases, the electrodes can generate one or more electric fields to, for example, stimulate organoid activity such as neural firing.10076] Unlike conventional multi-electrode array (MEA) that requires 2D cell culture and direct cell contacts on the surface of electrodes, the disclosed non-invasive real-time monitoring of ADRD-MAP (Alzheimer’s Disease and Related Dementias - Molecular Atlas Project) is based onnanostructured electrode-based Electroencephalography (EEG or nano-EEG) readouts. The disclosed systems and methods include non-contact field potential imaging of organ such as brain activities from 3D mini-brains. In some cases, the nano-EEG per chamber can include multichannel readouts in total through an array of contact pads organized along the substrate edge. In some cases, there are 1680 multi-channel readouts. In some cases, the monitoring system 100 can include multi-channel amplifiers, analog-to-digital converters (ADC), and contact springs on a head-stage printed circuit board. In some cases, an environmental Faraday cage can enclose the entire assembly for reduced electromagnetic noise and regulated temperature, CO2, and humidity.10O77| The fabrication of the electrodes can include a series of micromachining, electrochemical surface modification, and depositing graphene quantum dots. The nanoEEG electrodes can include a 70-140nm-thick platinum layer and a galvanic reaction that allows us to create a nanostructured electrode and gain improved sensitivity in low-frequency for detecting brain waves. The submerged electrodes can be electrically coupled to the external measurement system using a custom connector array. In some cases, the customer connector array includes 8-layer rigid-flex printed circuit board (PCB) soldered to an array of 384 laser micro-machined stainless steel leaf spring connectors. The rigid-flex PCB can be connected to a digital multiplex PCB containing switches and a microcontroller unit. Additionally or alternatively, the electrodes may double as perfusion tubes of the pumps.|0078| FIGS. 8A-E depict brainwave monitoring of MPP+-induced neurodegenerative progress in midbrain organoids. FIG. 8A illustrates the toxicokinetic impact of MPP+ on midbrain dopaminergic signaling. The Dopamine Transporter (DAT) controls extracellular dopamine levels in midbrain tissue. MPP+’s affinity for DAT can disrupt the extracellular clearance of dopamine, leading to abnormal excitation of dopamine-receiving populations. Internalized MPP+ within dopaminergic neurons progressively accumulates in mitochondria, disrupting the mitochondrial electron transport chain and causing ATP (adenosine triphosphate) depletion and eventual cell death.

[0079] FIG. 8B illustrates distinct EEG and LFP patterns and corresponding brainwaves before and during chronic MPP+ application. The MPP+ stages, labeled as "Early," "Middle," and "Late,"represent 3, 8, and 12 days of MPP+ perfusion, respectively, in this example. The early and middle stages can exhibit neuronal hyperactivity in the theta-to-gamma band, accompanied by increased spiking activity, which is evident in the EEG-LFP difference. The late stage is characterized by intermittent oscillations, marked by shorter and sporadic LFP bursts.[00801 FIG. 8C illustrates time-averaged brainwave power trends of midbrain organoids during MPP+ exposure. In this figure, organoid-average brainwaves (n=20) are normalized against baseline values, with shaded areas representing standard deviations. The hyperactivity features of this example peak around day 3 of MPP+ perfusion and gradually decrease to electrophysiological inactivity. The arrows denote the earliest time points at which a significant difference emerged between MPP+-treated and age-matched control organoids (n=20 for each group).[00811 FIG. 8D illustrates phase-amplitude coupling (PAC) analysis for brainwave alterations associated with MPP+ exposure, indicating abnormal delta-beta coupling during the MPP+- induced hyperactivity. Time-resolved traces of modulation index (MI) between the phase and amplitude frequency are computed at one-second intervals during the same time spans as the episodes in FIG. 8B. The time-resolved MI trace demonstrated that the delta-beta coupling correlated with LFP bursts during the hyperactive phase. Beta-silent transient intervals exhibited baseline MI levels during the hyperactivity. Notably, the synchronization between the increase in MI and LFP bursts, particularly beta rhythm bursts, did not correspond to the EEG patterns observed before the MPP+ application.

[0082] FIG. 8E depicts a temporal trace of sorted MI values during the MPP+ exposure. The time-resolved MI values are sorted and divided into the top and bottom 10% based on magnitude. By sorting the MI levels of individual organoid EEGs, transient increases in the top MI values during the hyperactive period are observed, while the bottom MI values remained consistent regardless of MPP+. Overall, the delta-beta PAC phenomenon was identified as a specific occurrence that synchronously emerged with beta rhythm during the hyperactive state induced by MPP+ in midbrain organoids.

[0083] Through the systems and methods described herein, high-density surface electrodes can be positioned in close proximity to organoids without direct contact. This configuration can enablethe capture of tissue-level electrical activity, distinct from conventional multi el ectrode arrays. Integrating electrical monitoring sensors and microfluidic perfusion controls allows for simultaneous organoid culture and observation. The system can support multiple organoids with parallel perfusion dynamics. It can perform independent biochemical modulations, while each organoid is monitored by non-contact electrodes for electroencephalogram (EEG)-like readout, with reference electrodes exposed to the upstream fresh perfusion. The miniaturized system design allows for MAP (Mitogen-Activated Protein) integration with multi-channel head stage amplifiers within a cell culture incubator. The eMPS (Engineered Microphy si ologi cal System) allows brain organoids to form on a chip and enables real-time mapping of brainwave dynamics using noncontact, non-invasive EEG. It creates an environment for brain tissue, supporting metabolic balance, electrophysiological activities, and neurotransmitter synthesis. Unlike traditional methods, the eMPS facilitates controlled perfusion, improving reliability and consistency of brain organoid production. The integrated EEG array allows real-time monitoring of brainwaves and detailed observation of developmental and pathological processes.Section C: Blood-Brain Barrier Modeling

[0084] Owing to its critical role in safeguarding the homeostasis of the brain’s microenvironment, it has long been postulated that alterations in the BBB may partake in brain diseases, including the cognitive dysfunction associated with aging. However, due to the lack of suitable methods to assess BBB integrity quantitatively and regionally in the human brain in vivo, it has been challenging to demonstrate disruptions of the BBB affecting brain regions involved in cognition.

[0085] To account for this and other challenges, the technical solutions proposed herein perturb BBB-permeability by targeting the tight junction protein, claudin-5 using CRISPR technology (c5- BBB-eMPS). The combination of nutrient perfusion and concurrent shear stress can enhance endothelial differentiation, tight junction protein expression, and reduce permeability, thereby better mimicking the human brain BBB. This 3D BBB microvascular model can aid in investigations towards how dynamic transport of small and large molecules across the BBBimpacts hippocampal development and function and how a compromised BBB permeability can potentially perturb this balance.(0086] In brief overview, the systems and methods described herein can enable 1.) Controlled transport and gradients of morphogens, growth factors, nutrients, oxygen, and monitoring of secreted factors such as neurotransmitters, 2.) Precise programmable flow and pressure control, 3.) Non-invasive continuous monitoring of brainwave activity during development and experimental perturbations, 4.) High number of replicates for robust experimental design and statistical power 5.) Open architecture for facile tissue introduction and removal, 6.) High magnification to non-invasively monitor tissues using confocal and epi-fluorescence imaging techniques over the course of neural development, 7.) Easy access to medias for sampling and offline analysis.(0087) Hippocampal networks display periodic network-driven events at frequencies of 0.13 ± 0.04 Hz (delta waves). Through the integration of the systems and methods described in Sections A and B, glutamatergic contributions to the dynamics of oscillatory waves and how they impact hippocampal neuronal networks can be determined.|0088| FIG. 9 displays an example 3D generation of an organoid. In some cases, the 3D BBB can be created by seeding on a soft ECM-like hydrogel (e.g., the membrane 115), endothelial cells (ECs) pericytes (PCs) and astrocytes (ACs) on a hard optically cleared bio-printed plastic carrier (e.g., the carrier 110) with embedded microfluidic channels (e.g., the channels 150) supporting perfusion. In some cases, the device (e.g., seeded membrane) can kept in an incubator for a period of time, such as 7 days (37C, 5% CO2). In some cases, the membrane can be seeded within the MPS. In some cases, medium can be replaced periodically, such as every 24 h. The generated 3D BBB model can be formed by a vasculogenesis-like process, giving rise to a well-connected and perfusable microvascular network (mVN) in a microfluidic device interacting via paracrine, juxtacrine and mechanical signaling.|0089| FIG. 9 further shows generation of an organoid such as a hippocampus. In a developing embryo, hippocampus can arise from medial pallium and lies between the neocortex and the dorsal midline structures including the choroid plexus and cortical hem. These two signaling centers canfunction as dorsaiizing organizers and can contribute to medial pallium patterning due to their secretion of WNT (Wingless and Inti) and BMP (Bone Morphogenetic Proteins) molecules. In some cases, to generate a hippocampus a culture condition for the dominant differentiation into the most dorsal region (neocortex) is defined. In some cases, intermediate region tissues: cortical hem, choroid plexus and medial pallium are induced.

[0090] In some cases, generating the hippocampus organoid can include l)seeding a set of cells into the retaining ring on top of the BBB ( which, in some cases can have previously seeded astrocytes, pericytes and endothelial cells in the lumen of the 3D-sNV) to perfuse the hippocampus, 2) delivering via microfluidics WNT and BMP signals between DO and D21 of hippocampal culture, to mimic hippocampal development and induce its patterning, 3) adding additional materials such as small molecules including AA (Arachidonic Acid), cAMP (Cyclic Adenosine Monophosphate) and BDNF (Brain-Derived Neurotrophic Factor) to the culture to enhance maturation. These adjustments will provide a better diffusion of oxygen, nutrients and patterning molecules to support formation of larger, continuous brain regions with myelinating cells.

[0091] The systems and method described herein can be utilized to test a variety of conditions of brain organoids and the BBB. For example, the disclosed technical solutions can enable the exploration of BBB permeability and determining its effects on hippocampal aging: a claudin5 KO. In some cases, 3D BBB permeability can be perturbed via CRISPR-CAS9 technology by introducing mutations of claudin-5 tight junction protein into iPSCs obtained from healthy patients. Accounting for a potential BBB disruption, CSF (Cerebrospinal Fluid) biomarkers of BBB permeability and vascular damage along with neuronal markers can be examined using the MPS system described herein.|0092| Using the systems and methods described herein, the BBB can be tested for changes to enzymes related to BBB disruption i.e.: metalloproteinases (Mmp9), potentially elevated endothelial tumor necrosis factor alpha (TNFa), Pericyte marker PDGFRB (Platelet-Derived Growth Factor Receptor Beta).

[0093] BBB compromised permeability can be tested on overall hippocampal development and health using the systems and methods described herein. A systematic analysis can explore: A) overall size and morphology by quantifying volume and surface area; B) cell proliferation and migration by testing if neural progenitor cells follow a stereotyped organization with a glutamatergic neurogenesis sequence (i.e.: Pax6^Ngn2^Tbr2^NeuroD^Tbrl ). The Wnt pathway can be required to promote neural progenitor cells’ sternness, its signaling can be compared across groups and their control organoids; C) the ability to generate hippocampal neuronal diversity, by following marker expression and comparing the developmental time course of neurons in distinct regions (i.e.: KAl’s CA3 vs Proxl’s DG); D) derived HO’s neuronal oscillations as an endophenotype for cognitive processing compared to control BBB-eMPS, among others.

[0094] Through the systems and methods described herein, a healthy and diseased BBB can be generated and analyzed for its impact in (1) oscillatory network dynamics, (2) molecular basis of oscillatory activity via studying excreted neurotransmitter, (3) phenomena of hippocampal neurogenesis and aging. Specifically, the system includes an MCP and synthetic carrier capable of enabling a perfusable BBB-neural organoid. The precision micropumps, in combination with the bio printed materials and geometry, enable exceptional control over the tissue’s mechanical environment, as well as the biochemical environment.|0095| The processes, methods, or algorithms disclosed herein can be deliverable to / implemented by a processing device, controller, or computer, which can include any existing programmable electronic control unit or dedicated electronic control unit. Similarly, the processes, methods, or algorithms can be stored as data and instructions executable by a controller or computer in many forms including, but not limited to, information permanently stored on non- writable storage media such as read-only memory (ROM) devices and information alterably stored on writable storage media such as floppy disks, magnetic tapes, compact discs (CDs), RAM devices, and other magnetic and optical media. The processes, methods, or algorithms can also be implemented in a software executable object. Alternatively, the processes, methods, or algorithms can be embodied in whole or in part using suitable hardware components, such as Application Specific Integrated Circuits (ASICs), Field-Programmable Gate Arrays (FPGAs), state machines,controllers or other hardware components or devices, or a combination of hardware, software and firmware components.(0096] While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms encompassed by the claims. The words used in the specification are words of description rather than limitation, and it is understood that various changes can be made without departing from the spirit and scope of the disclosure. As previously described, the features of various embodiments can be combined to form further embodiments of the invention that may not be explicitly described or illustrated. While various embodiments could have been described as providing advantages or being preferred over other embodiments or prior art implementations with respect to one or more desired characteristics, those of ordinary skill in the art recognize that one or more features or characteristics can be compromised to achieve desired overall system attributes, which depend on the specific application and implementation. These attributes can include, but are not limited to strength, durability, life cycle, marketability, appearance, packaging, size, serviceability, weight, manufacturability, ease of assembly, etc. As such, to the extent any embodiments are described as less desirable than other embodiments or prior art implementations with respect to one or more characteristics, these embodiments are not outside the scope of the disclosure and can be desirable for particular applications.10097] With regard to the processes, systems, methods, heuristics, etc. described herein, it should be understood that, although the steps of such processes, etc. have been described as occurring according to a certain ordered sequence, such processes could be practiced with the described steps performed in an order other than the order described herein. It further should be understood that certain steps could be performed simultaneously, that other steps could be added, or that certain steps described herein could be omitted. In other words, the descriptions of processes herein are provided for the purpose of illustrating certain embodiments and should in no way be construed so as to limit the claims.[00981 Accordingly, it is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments and applications other than the examples provided would be apparent upon reading the above description. The scope should be determined, not withreference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. It is anticipated and intended that future developments will occur in the technologies discussed herein, and that the disclosed systems and methods will be incorporated into such future embodiments. In sum, it should be understood that the application is capable of modification and variation.

[0099] All terms used in the claims are intended to be given their broadest reasonable constructions and their ordinary meanings as understood by those knowledgeable in the technologies described herein unless an explicit indication to the contrary is made herein. In particular, use of the singular articles such as “a,” “the,” “said,” etc. should be read to recite one or more of the indicated elements unless a claim recites an explicit limitation to the contrary.

[0100] The abstract of the disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various embodiments for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

Claims

WHAT IS CLAIMED IS:

1. A neural microphysiological system for simulating a blood brain barrier, comprising: a culture plate configured to receive at least one neural model; the at least one neural model including a carrier adj acent to at least one pair of wells, the wells configured to receive one or more tissues and be fluidly connected with the carrier; a membrane coupled to the carrier and configured to receive a set of cells and enable perfusion throughout the membrane.

2. The system of claim 1, further comprising a lid including micropumps configured to provide materials to the membrane.

3. The system of claim 2, wherein the carrier defines at least one channel configured to deliver materials to the membrane.

4. The system of claim 1, wherein the at least one pair of wells includes two pairs of wells, one of each pair of the respective wells arranged on opposite sides of the carrier.

5. The system of claim 1, wherein the carrier includes a vasculature and retaining ring arranged at an opening defined by the carrier.

6. The system of claim 1, wherein the carrier is configured to couple to a microphysiological platform.

7. The system of claim 1, wherein the carrier defines at least two channels to generate a first fluid flow to the membrane and a second, separate fluid flow to the membrane.

8. The system of claim 1, wherein the carrier is formed by bio-ink.

9. The system of claim 1, wherein the membrane is formed via two-photon printing.

10. The system of claim 1 , wherein the carrier is configured to maintain a neural organoid.

11. The system of claim 1, comprising a lid including an electrode array to monitor an organoid.

12. The system of claim 11, wherein the culture plate is configured to receive a plurality of the at least one neural models and the lid encapsulates the neural models between the lid and the culture plate.

13. A neural model for a microphysiological system for simulating a blood brain barrier comprising: a membrane including a structure arranged to enable perfusion throughout the membrane; and a carrier configured to receive the membrane and defining at least one channel coupled to reservoir wells housing tissue, the carrier configured to receive and maintain a neural organoid via the delivery of cells via the membrane.

14. The model of claim 13, wherein the carrier is configured to be received by a culture plate and housed between the culture plate and a lid.

15. The model of claim 14, wherein the reservoir wells are configured to transmit tissue via the channels to the membrane via micropumps arranged in the lid.

16. The model of claim 15, wherein the reservoir wells include at least one pair of wells, each well of the pair arranged on opposite sides of the carrier.

17. The model of claim 15, wherein the neural organoid is monitored by electrodes arranged in the lid.

18. The model of claim 13, wherein the carrier includes a vasculature and retaining ring arranged at an opening defined by the carrier to facilitate delivery of materials to and from the membrane.

19. The model of claim 13, wherein the at least one channel includes two channels to generate a first fluid flow to the membrane and a second, separate fluid flow to the membrane.

20. The model of claim 18, wherein the membrane is formed via two-photon printing.

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