Brain-assessment devices and methods of making and using the same

Abstract

Brain-Assessment devices are provided. Aspects of the devices include a brain matrix chamber comprising a brain matrix composition. In embodiments, the brain matrix composition includes a three-dimensional endothelial cell vascular network (e.g., comprising brain microvascular endothelial cells) and a cellular component comprising one or more of brain parenchymal cells and peripheral immune cells. Systems comprising a device of the present disclosure and a reader (e.g., comprising an imaging system) are also provided. Methods of preparing a device of the present disclosure for use in an assay are provided. Also provided are methods of introducing a perturbation (e.g., a small molecule) to a device of the present disclosure.

Description

BRAIN-ASSESSMENT DEVICES AND METHODS OF MAKING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application serial no. 63 / 637,068, filed on April 22, 2024, which is incorporated by reference herein in its entirety.INTRODUCTION

[0002] Healthy cognition depends on intact function and homeostasis of brain parenchymal cells, peripheral immune cells, and other factors that may deteriorate with age and / or neurodegenerative diseases. There is a need for therapeutic interventions that can halt or restore declining cognitive function associated with age and / or neurodegenerative disease.

[0003] However, existing approaches for assessing potential therapeutic interventions suffer from poor translatability in the clinic. In vivo animal models have long been considered the standard for pre-clinical evaluation of therapeutic interventions but offer low reproducibility with a >95% clinical trial failure rate. While such existing in vivo approaches have physiological relevance, this advantage is undermined due to fundamental species-specific differences between animal models and humans. Additionally, existing in vivo approaches offer low throughput and high variability, increasing time and costs for evaluating therapeutic interventions.

[0004] On the other hand, existing in vitro approaches (e.g., 2D cell cultures) offer better reproducibility but fail to capture key aspects of brain physiology that are necessary for successful translatability. For example, 2D in vitro cell cultures lack the physiologically relevant three-dimensional structures and microenvironments of in vivo tissues. While static 3D in vitro cell cultures provide a three-dimensional environment for cell growth, such approaches still fail to fully recapitulate key aspects, such as fluid flows, of the physiology of in vivo tissues.SUMMARY

[0005] The inventors have realized that there is a need for compositions and methods to reproducibly model relevant brain physiology and enable the discovery of clinically translatable interventions for restoring cognitive function. Brain- Assessment devices are provided. Aspects of the devices include a brain matrix chamber comprising a brain matrix composition. In embodiments, the brain matrix composition includes a three- dimensional endothelial cell vascular network (e.g., comprising brain microvascular endothelial cells) and a cellular component comprising one or more brain parenchymal cells, e.g., astrocytes, pericytes, neurons, microglia, and oligodendrocytes. In some instances, lumens of the three-dimensional endothelial cell vascular network are filled with a fluid that includes peripheral immune cells. In other instances, assemblies are provided where a source of fluid comprising peripheral immune cells is fluidically coupled to a lumen of the three-dimensional endothelial cell vascular network. Systems comprising a device or assembly of the present disclosure and a reader (e.g., comprising an imaging system) are also provided. Methods of preparing a device or assembly of the present disclosure for use in an assay are provided. Also provided are methods of introducing a perturbation (e.g., a small molecule) to a device or assembly of the present disclosure.

[0006] The present disclosure provides assemblies and devices comprising a brain matrix chamber comprising a brain matrix composition, wherein the brain matrix composition comprises a three-dimensional endothelial cell vascular network and a cellular component comprising one or more of brain parenchymal cells, e.g., astrocytes, pericytes, neurons, microglia and oligodendrocytes. In some cases, the three-dimensional endothelial cell vascular network comprises brain microvascular endothelial cells. In some cases, the three-dimensional endothelial cell vascular network comprises human umbilical vein endothelial cells. In some embodiments, the human umbilical vein endothelial cells overexpress ETV2. In some embodiments, the three-dimensional endothelial cell vascular network comprises lumens having an average diameter ranging from 5pm to 100pm. In some cases, the neurons are external to lumens of the three-dimensional endothelial cell vascular network. In some cases, at least some of the neurons are differentiated from induced pluripotent stem cells. In some embodiments, the brain matrix composition further comprises astrocytes. In some cases, the astrocytes are external to lumens of thethree-dimensional endothelial cell vascular network. In some cases, at least some of the astrocytes are directly associated with the three-dimensional endothelial cell vascular network. In some cases, at least some of the astrocytes are directly associated with neurons. In some embodiments, the astrocytes are derived from fibroblasts. In some embodiments, the astrocytes are differentiated from induced pluripotent stem cells. In some embodiments, the brain matrix composition further comprises microglia. In some cases, the microglia are external to lumens of the three-dimensional endothelial cell vascular network. In some cases, at least some of the microglia are directly associated with neurons. In some cases, at least some of the microglia are directly associated with astrocytes. In some cases, at least some of the microglia are directly associated with the three-dimensional endothelial cell vascular network. In some embodiments, the microglia are differentiated from induced pluripotent stem cells. In some embodiments, the brain matrix composition further comprises pericytes. In some cases, the pericytes are external to the lumens of the three-dimensional endothelial cell vascular network. In some cases, at least some of the pericytes are directly associated with astrocytes. In some embodiments, the brain matrix composition further comprises oligodendrocytes. In some cases, the oligodendrocytes are external to the lumens of the three-dimensional endothelial cell vascular network. In some cases, at least some of the oligodendrocytes are directly associated with neurons. In some cases, at least some of the oligodendrocytes are directly associated with astrocytes. In some cases, at least some of the oligodendrocytes are directly associated with microglia.

[0007] In some instances, lumens of the three-dimensional endothelial cell vascular network are filled with a fluid that includes peripheral immune cells. In other instances, assemblies are provided where a source of fluid comprising peripheral immune cells is fluidically coupled to a lumen of the three-dimensional endothelial cell vascular network.

[0008] In some embodiments, the brain matrix composition comprises a gel. In some embodiments, the gel is a hydrogel. In some cases, the gel comprises one or more polymers. In some embodiments, one or more of the polymers are proteins. In some cases, the proteins comprise fibrin. In some embodiments, one or more of the polymers are polysaccharides. In some embodiments, the hydrogel stiffness ranges from lOOPa to lOOOPa. In some embodiments, the microfluidic device further comprises a first perfusion channel fluidically coupled to the brain matrix chamber. In some embodiments, themicrofluidic device further comprises a second perfusion channel fluidically coupled to the brain matrix chamber. In some cases, the first and second perfusion channels are separated from the brain matrix chamber by a boundary wall. In some cases, the boundary wall is less than the height of the perfusion channel.

[0009] In some embodiments, the lumens of the three-dimensional endothelial cell vascular network are filled with fluid. In some embodiments, the fluid has an average flow velocity in the lumens ranging from O.lmm / sec to lOmm / sec. In some embodiments, the fluid has a total volumetric flow rate in the brain matrix composition in the range of 10ml / (100g min) to 100ml / (100g min). In some embodiments, the fluid has a pressure in the range of 1mm Hg to 20mm Hg. In some cases, the fluid is aqueous. In some embodiments, the fluid comprises T cells. In some embodiments, the fluid further comprises monocytes. In some embodiments, the fluid further comprises blood components. In some cases, the blood components further comprise B cells.

[0010] In some embodiments, the brain matrix composition is stable for one week or longer. In some cases, the brain matrix composition is stable for two weeks or longer. In some cases, the brain matrix composition is stable for three weeks or longer. In some cases, the brain matrix composition is stable for four weeks or longer.

[0011] The present disclosure provides systems comprising a device or assembly of the present disclosure and a reader. In some embodiments, the reader comprises an imaging system. In some cases, the imaging system comprises a magnification device. In some cases, the imaging system comprises an image sensor. In some cases, the imaging system comprises a light source. In some cases, the imaging system is configured to capture images of the brain matrix chamber. In some embodiments, the system further comprises an environmental control system. In some cases, the environmental control system comprises a temperature control system. In some cases, the environmental control system comprises an atmospheric control system. In some embodiments, the system further comprises a fluid control system. In some cases, the fluid control system comprises a fluid actuator. In some cases, the fluid control system further comprises a flow sensor. In some cases, the fluid control system further comprises a pressure sensor. In some embodiments, the fluid control system is fluidically coupled to the brain matrix chamber. In some cases, the fluid control system can modulate the total volumetric flow rate of a fluid through the brain matrix composition. In some embodiments, the total volumetricflow rate of the fluid is in the range of 1 microliter / min to 10 ml / min through the perfusion chambers. In some cases, the fluid control system can modulate the flow velocity of a fluid in the lumens of the three-dimensional endothelial cell vascular network. In some embodiments, the flow velocity of the fluid is in the range of O. lmm / sec to lOmm / sec. In some cases, the fluid control system can modulate the pressure of a fluid in the lumens of the three-dimensional endothelial cell vascular network. In some embodiments, the pressure of the fluid is in the range of 1mm Hg to 20mm Hg.

[0012] The present disclosure provides methods of preparing a device of the present disclosure for use in an assay, the method comprising introducing cells into the lumens of the three-dimensional endothelial cell vasculature. In some cases, the cells comprise T cells. In some cases, the cells further comprise monocytes. In some embodiments, the methods further comprise introducing blood components into the lumens of the three- dimensional endothelial cell vascular network. In some cases, the blood components further comprise B cells.

[0013] The present disclosure provides methods of introducing a perturbation to a device of the present disclosure. In certain embodiments the perturbation may comprise a small molecule, a nucleic acid, a peptide, a nanoparticle, a viral vector, a non-viral vector, a virus, a cell, a mechanical stress and / or a change in temperature. In some embodiments, the methods further comprise imaging the brain matrix composition. In some cases, the methods further comprise fixing the brain matrix composition. In some cases, the methods further comprise permeabilizing the brain matrix composition. In some cases, the methods further comprise immunostaining the brain matrix composition. In some embodiments, the methods further comprise harvesting at least a portion of the brain matrix composition. In some cases, the methods further comprise dissociating the brain matrix composition. In some embodiments, the methods further comprise lysing a cell of the brain matrix composition. In some embodiments, the methods further comprise isolating a nucleic acid from the cell. In some cases, the methods further comprise sequencing the nucleic acid. In some cases, the methods further comprise measuring the expression level of the nucleic acid. In some embodiments, the methods further comprise isolating a protein from the cell. In some cases, the methods further comprise identifying the protein by mass spectrometry. In some cases, the methods further compriseidentifying a post-translational modification of the protein. In some cases, the methods further comprise measuring the expression level of the protein. In some embodiments the methods further comprise isolating a metabolite from the cell. In some cases, the methods further comprise isolating identifying the metabolite by mass spectrometry. In some cases, the methods further comprise measuring the abundance of the metabolite. In some embodiments, the methods further comprise isolating a lipid from the cell. In some cases, the methods further comprise identifying the lipid by mass spectrometry. In some cases, the methods further comprise measuring the abundance of the lipid.

[0014] The present disclosure additionally provides kits comprising a device of the present disclosure. In some embodiments, the kits further comprise a population of T cells. In some embodiments, the kits further comprise a population of monocytes. In some embodiments, the kits further comprise blood components. In some cases, the blood components further comprise B cells. In some embodiments, the kits further comprise a fresh media.BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1A depicts an exemplary perfuse-able three-dimensional endothelial cell vascular network. FIG. IB illustrates example z-stack (above) through the vasculature in a brain-matrix with perfusion of T cells. Some T cells can be seen attached to the lumen of the vasculature in z=40um plane, while a T cell can be seen flowing through the lumen in z=60um plane. While observing the brain-matrix over time (FIG. 1C), a T cell can be seen extravasating from the vascular lumen to the abluminal space. Images were taken on a confocal microscope at 20x magnification.

[0016] FIGs. 2A-2D depict exemplary schema for obtaining astrocytes. A) depicts a schema for isolating and culturing primary astrocytes. B) depicts primary astrocytes successfully isolated and cultured from mouse brain tissue. C) depicts a schema for differentiation of iPSCs to astrocytes. D) depicts a schema for direct conversion of fibroblasts to astrocytes.

[0017] FIG. 3 depicts differentiation of induced pluripotent stem cells to microglia.

[0018] FIGs. 4A-4C depict a representative microfluidic device of the present disclosure. A) depicts a top-down view schematic of an exemplary microfluidic device. B) depicts a top-down view image of a representative microfluidic device. C) depicts a transversecross-sectional view schematic of the center of a representative microfluidic device. FIGs. 4D-4E depict another representative microfluidic device of the present disclosure. D) depicts a top-down view schematic of a representative microfluidic device. E) depicts a transverse cross-sectional view schematic of the center of a representative microfluidic device.

[0019] FIG. 5 depicts an exemplary system comprising a microfluidic device and reader comprising an imaging system, fluid control system, and environmental control system.

[0020] FIG. 6 depicts a schematic of an exemplary microfluidic device of the present disclosure, methods of preparing a microfluidic device for an assay, and methods of introducing a perturbation to a microfluidic device.DETAILED DESCRIPTION

[0021] Brain- Assessment devices are provided. Aspects of the devices include a brain matrix chamber comprising a brain matrix composition. In embodiments, the brain matrix composition includes a three-dimensional endothelial cell vascular network (e.g., comprising brain microvascular endothelial cells) and a cellular component comprising one or more brain parenchymal cells, e.g., astrocytes, pericytes, neurons, microglia and oligodendrocytes. In some instances, lumens of the three-dimensional endothelial cell vascular network are filled with a fluid that includes peripheral immune cells. In other instances, assemblies are provided where a source of fluid comprising peripheral immune cells is fluidically coupled to a lumen of the three-dimensional endothelial cell vascular network. Systems comprising a microfluidic device of the present disclosure and a reader (e.g., comprising an imaging system) are also provided. Methods of preparing a microfluidic device of the present disclosure for use in an assay are provided. Also provided are methods of introducing a perturbation (e.g., a small molecule) to a microfluidic device of the present disclosure. The present disclosure additionally provides kits comprising a microfluidic device of the present disclosure.

[0022] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0023] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0024] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0026] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0027] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such,this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0028] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0029] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.BRAIN-ASSESSMENT DEVICES

[0030] Brain-assessment devices of the present disclosure are devices that may be used to assess the effect of a perturbation, e.g., a candidate therapeutic agent, on brain function. Devices of embodiments of the invention include a brain matrix chamber comprising a brain matrix composition, where fluidic inlet(s) and outlet(s) to the brain matrix chamber may be provided to allow introduction of components, e.g., cells, candidate therapeutic agents, etc., into the brain matrix composition. In some instances, the devices are microfluidic devices. Various components of the devices are now described in greater detail.Brain Matrix Composition

[0031] As reviewed in greater detail below, the brain matrix composition comprises a three-dimensional endothelial cell vascular network and a cellular component comprising one or more brain parenchymal cells, e.g., astrocytes, pericytes, neurons, microglia, and oligodendrocytes. In some instances, the lumens of the three-dimensional endothelial cellvascular network may be filled with a fluid comprising peripheral immune cells (e.g., T cells, monocytes) and / or other blood components. In other instances, one or more lumens of the three-dimensional endothelial cell vascular network may be fluidically coupled to a fluid comprising peripheral immune cells (e.g., T cells, monocytes) and / or other blood components. A brain matrix composition of the present disclosure may recapitulate aspects of brain function relevant for assessing a perturbation of interest. For example, the three-dimensional endothelial cell vascular network, in combination with pericytes and astrocytes of the brain matrix composition, may recapitulate the physiology of the bloodbrain barrier (BBB) which regulates the accessibility of agents in the blood stream to the brain. As another example, the brain matrix composition may recapitulate physical and / or cell-signaling interactions between neurons and glial cells (e.g., astrocytes, microglia, oligodendrocytes). As another example, the brain matrix composition may recapitulate physical and / or cell-signaling interactions between glial cells and the three-dimensional endothelial cell vascular network. As another example, a brain matrix composition may also recapitulate aspects of the physiology (e.g., the blood-brain barrier, neuron-glial interactions) of an aging brain. As another example, the flow of peripheral immune cells through the lumens of the three-dimensional endothelial cell vascular network may recapitulate features of neuroimmune interactions that have been implicated in neurodegenerative diseases and aging. In some cases, the cells of the brain matrix composition may model or recapitulate an aging or neurodegenerative disease phenotype.Endothelial Cell Vascular Network

[0032] A three-dimensional endothelial cell vascular network of the present disclosure may be comprised of lumenized (i.e., possessing a lumen, a hollow space within a tubular structure) tubular vessels comprised of endothelial cells. The tubular vessels of the vascular network may bifurcate into branching vessels and at least some of the vessels may interconnect with one another. The endothelial cells of the tubular vessel may be joined together by junctional complexes such as adherens junctions and / or tight junctions and surrounded by basement membrane proteins. Tight junctions are a type of endothelial cell-cell junction protein complex present across vertebrates that form a barrier to diffusion of substances through the intercellular spaces between endothelial cells. The tight junction protein complex may include: claudins (e.g., claudin-1), occludin, zonula occludens-1 (ZO-1), and junctional adhesion molecules (JAMs). Adherens junctions areanother type of cell-cell junction protein complex that function in regulation of the actin cytoskeleton, intracellular signaling, and transcriptional regulation. The adherens junction protein complex may include: E-cadherin, and various catenins (e.g., P-catenin). The basement membrane extracellular matrix (ECM) provides scaffolding and support for associated endothelial cells. Basement membrane extracellular matrix (ECM) proteins may include laminin and type IV collagen.

[0033] A subject three-dimensional endothelial cell vascular network of the present disclosure may allow passage or flow of a fluid through the lumens of the three- dimensional endothelial cell vascular network (i.e., perfusion of the vascular network). In some embodiments, a subject three-dimensional endothelial cell vascular network of the present disclosure comprises lumens having an average diameter from 5pm to 100pm, e.g., in some cases a subject three-dimensional endothelial cell vascular network can comprise lumens having an average diameter from 10pm to 90pm, such as from 20pm to 80pm, such as from 30pm to 70pm, and include from 40pm to 60pm. An exemplary embodiment of a three-dimensional endothelial cell vascular network is shown in FIG.1 A, depicting human umbilical vein endothelial cells (HUVECs; green), co-cultured with hTERT immortalized human astrocytes (red). The HUVECs are organized into a perfuseable three-dimensional endothelial cell vascular network in a fibrin gel scaffold. The lumens of the three-dimensional endothelial cell vascular network are ~50-70pm in diameter. A subject three-dimensional endothelial cell vascular network of the present disclosure may exhibit a reduced permeability to select molecules or substances compared to a two-dimensional endothelial cell monolayer. The permeability of a subject three-dimensional endothelial cell vascular network may be expressed as a permeability coefficient that describes the flux of colored solutes across the lumens of the vascular network to the exterior of the vascular network. Methods of measuring the permeability coefficients are described, for example, in Curry, F. E., V. H. Huxley, and R. H. Adamson. "Permeability of single capillaries to intermediate-sized colored solutes." American Journal of Physiology-Heart and Circulatory Physiology 245.3 (1983): H495-H505, the entirety of which is incorporated herein by reference.

[0034] FIG. IB illustrates example z-stack (above) through the vasculature in a brainmatrix with perfusion of T cells. Some T cells can be seen attached to the lumen of the vasculature in z=40um plane, while a T cell can be seen flowing through the lumen inz=60um plane. While observing the brain-matrix over time (FIG. 1C), a T cell can be seen extravasating from the vascular lumen to the abluminal space. Images were taken on a confocal microscope at 20x magnification.

[0035] Suitable endothelial cells for use in a subject three-dimensional endothelial cell vascular network of the present disclosure may include any endothelial cell capable of vascularization (i.e., forming lumenized tubular vessels). Suitable endothelial cells may be primary endothelial cells isolated from an in vivo source or may be differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, suitable endothelial cells may be primary human dermal microvascular endothelial cells (HDMVECs) isolated from blood vessels of the skin. Isolation and culture of HDMVECS is described, for example, in Bourland, Jennifer, et al. "Isolation and culture of human dermal microvascular endothelial cells." Skin Tissue Engineering: Methods and Protocols (2019): 79-90, the entirety of which is incorporated herein by reference. In some embodiments, suitable endothelial cells may be human umbilical vein endothelial cells (HUVECs) isolated from the umbilical cord. HUVECs are a widely used source of endothelial cells for in vitro studies of angiogenesis. Isolation and culture of HUVECs is described, for example, in Baudin, Bruno, et al. "A protocol for isolation and culture of human umbilical vein endothelial cells." Nature protocols 2.3 (2007): 481-485, the entirety of which is incorporated herein by reference. In some cases, HUVECs may be immortalized (i.e., can divide and proliferate indefinitely). As an example, HUVECs may be immortalized by expression of telomerase reverse transcriptase (TERT). Immortalization through expression of TERT is described, for example, at: www(dot)atcc(dot)org / resources / culture-guides / htert-immortalized-cell-culture- guide#Telomeres, the entirety of which is incorporated herein by reference. In some cases, HUVECs may over-express ETS variant transcription factor 2 (ETV2). ETV2 is a pioneer transcription factor for the development of cardiac, endothelial and hematopoietic cell lineages and its over-expression can regulate and reprogram endothelial cell development. In some embodiments, suitable endothelial cells may be brain microvascular endothelial cells (BMECs) isolated from brain tissue and key constituents of the blood brain barrier (BBB). Isolation and culture of BMECs is described, for example, in Navone, Stefania E., et al. "Isolation and expansion of human and mousebrain microvascular endothelial cells." Nature protocols 8.9 (2013): 1680-1693, the entirety of which is incorporated herein by reference.

[0036] In some embodiments, the lumens of a subject three-dimensional endothelial cell vascular network are filled with fluid. The average rate at which the fluid flows (i.e., flow velocity) through the lumens of three-dimensional endothelial cell vascular network may vary. In some embodiments, the fluid has an average flow velocity from O.lmm / sec to lOmm / sec, e.g., in some cases the average flow velocity is in the range from 0.5mm / sec to 9.5mm / sec, such as from Imm / sec to 9mm / sec, such as from 1.5mm / sec to 8.5mm / sec, such as from 2mm / sec to 8mm / sec, such as from 2.5mm / sec to 7.5mm / sec, such as from 3mm / sec to 7mm / sec, such as from 3.5mm / sec to 6.5mm / sec, such as from 4mm / sec to 6mm / sec, and including from 4.5mm / sec to 5.5mm / sec. The flow of the fluid through the brain matrix composition may also be described by the volumetric flow rate of the fluid. The total volumetric flow rate may be described as the volume of fluid that passes through a given mass of brain matrix composition per unit time, e.g., ml / (g min). In some embodiments, the fluid has a volumetric flow rate ranging from 10ml / (100g min) to 100ml / (100g min), e.g., in some cases the volumetric flow rate is in the range from 10ml / (100g min) to 90ml / (100g min), such as from 20ml / (100g min) to 80ml / (100g min), such as from 30ml / (100g min) to 70ml / (100g min), and including from 40ml / (100g min) to 60ml / (100g min). The pressure of the fluid in the lumens of a subject three- dimensional endothelial cell vascular network (i.e., the perfusion pressure) may vary. In some embodiments, the fluid has a pressure in the range from 1mm Hg to 20mm Hg, e.g., in some cases the pressure is in the range from 2mm Hg to 19mm Hg, such as from 3mm Hg to 18mm Hg, such as from 4mm Hg to 17mm Hg, such as from 5mm Hg to 16mm Hg,such as from 6mm Hg to 15 mm Hg, such as from 7mm Hg to 14mm Hg, such as from 8mm Hg to 13 mm Hg, such as from 9mm Hg to 12mm Hg, and including from 10mm Hg to 11mm Hg. In some cases, the pressure of the fluid may drive flow of the fluid through the lumens of a subject three-dimensional endothelial cell vascular network. In some embodiments, the fluid may have any combination of the attributes discussed above. In some cases, the fluid is aqueous. In some cases, the fluid is a cell growth medium (e.g., endothelial growth medium, Dulbecco’s modified eagle medium, human plasma-like medium).Neurons

[0037] In some embodiments, the neurons of a subject brain matrix composition are external to the lumens of a subject three-dimensional endothelial vascular network (i.e., the neurons are present on the abluminal side of the three-dimensional endothelial vascular network). Suitable neurons for use in a subject brain matrix composition may be primary neurons isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the neurons may be differentiated from iPSCs. Methods for differentiating iPSCs into neurons are known in the art and described, for example, in Bell, Scott, et al. "Differentiation of human induced pluripotent stem cells (iPSCs) into an effective model of forebrain neural progenitor cells and mature neurons." Bio-protocol 9.5 (2019): e3188-e3188, the disclosure of which is incorporated herein by reference.Pericytes

[0038] In some embodiments, a subject brain matrix composition may further comprise pericytes. Pericytes are key components of the BBB and are known to play roles in regulating BBB permeability, hemodynamic responses, and angiogenesis, among others. In some cases, the pericytes are external to the lumens of a subject three-dimensional endothelial vascular network (i.e., the lumens are present on the abluminal side of the three-dimensional endothelial vascular network). In some embodiments, at least some of the pericytes are directly associated with the three-dimensional endothelial vascular network of a subject brain matrix composition. Suitable pericytes for use in a subject brain matrix composition may be primary pericytes isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some cases, the pericytes may be primary pericytes isolated from microvasculature of a tissue (e.g., microvasculature of placental tissue). In some embodiments, the pericytes are differentiated from induced pluripotent stem cells (iPSCs). For example, iPSCs may be differentiated to pericytes by modulation of Wnt / p-catenin signaling and treatment with pericyte growth factors (e.g., PDGF-BB). Methods for isolating primary pericytes, generating pericytes from iPSCs, and culturing pericytes are described, for example, in Alvino, Valeria Vincenza, et al. "Approaches for the isolation and long-term expansion of pericytes from human andanimal tissues." Frontiers in Cardiovascular Medicine 9 (2023): 1095141, the entirety of which is incorporated herein by reference.Astrocytes

[0039] In some embodiments, a subject brain matrix composition may further comprise astrocytes. Astrocytes are known to play a role in various brain functions such as synaptogenesis, regulating BBB permeability, and maintaining extracellular homeostasis. In some cases, the astrocytes are external to the lumens of a subject three-dimensional endothelial vascular network (i.e., the astrocytes are present on the abluminal side of the three-dimensional endothelial vascular network). In some embodiments, at least some of the astrocytes are directly associated with the three-dimensional endothelial vascular network of a subject brain matrix composition. In some cases, at least some of the astrocytes may extend processes, known as endfeet, that make direct contact with the three-dimensional endothelial vascular network of a subject brain matrix composition. In some embodiments, at least some of the astrocytes are directly associated with the pericytes of a brain matrix composition. In some cases, at least some of the astrocytes may extend endfeet that make direct contact with the pericytes of a subject brain matrix composition. In some embodiments, at least some of the astrocytes are directly associated with the neurons of a subject brain matrix composition. In some cases, at least some of the astrocytes may extend processes that make direct contact with dendrites or synapses of the neurons of a subject brain matrix composition. Suitable astrocytes for use in a subject brain matrix composition may be primary astrocytes isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the astrocytes may be primary astrocytes isolated from central nervous system tissues. FIG. 2A depicts a schema of an exemplary embodiment for isolating and culturing primary astrocytes. FIG. 2B depicts primary astrocytes successfully isolated and cultured from mouse brain tissue (immunostained for glial fibrillary acidic protein; nuclear stained with DAPI). Isolation and culture of primary astrocytes is described, for example, in Jiang, Lu-Lin, et al. "Membralin deficiency dysregulates astrocytic glutamate homeostasis, leading to ALS-like impairment." The Journal of Clinical Investigation 129.8 (2019): 3103-3120, the entirety of which is incorporated herein by reference. In some embodiments, the astrocytes are differentiated from induced pluripotent stem cells (iPSCs). FIG. 2C depicts a schema of an exemplary embodimentfor differentiation of iPSCs to astrocytes. Methods for differentiating iPSCs into astrocytes are described, for example, in Leventoux, Nicolas, et al. "Human astrocytes model derived from induced pluripotent stem cells." Cells 9.12 (2020): 2680, the entirety of which is incorporated herein by reference. In some embodiments, the astrocytes may be derived from fibroblasts. In some cases, the astrocytes may be directly converted from fibroblasts by expression of SRY-box transcription factor 9 (SOX9), nuclear factor I A (NFIA), and nuclear factor I B (NFIB) in fibroblasts. FIG. 2D depicts a schema of an exemplary embodiment for direct conversion of fibroblasts to astrocytes. Further details regarding direct conversion of fibroblasts into astrocytes can be found in Quist, Ella, et al. "Transcription factor-based direct conversion of human fibroblasts to functional astrocytes." Stem Cell Reports 17.7 (2022): 1620-1635, the entirety of which is incorporated herein by reference.Microglia

[0040] In some embodiments, a subject brain matrix composition may further comprise microglia. Microglia are known to play a role in brain development and function, including phagocytosis, and regulating neural circuits through pruning of synapses. In some cases, the microglia are external to the lumens of a subject three-dimensional endothelial vascular network (i.e., the microglia are present on the abluminal side of the three-dimensional endothelial vascular network). In some embodiments, at least some of the microglia are directly associated with the neurons of a subject brain matrix composition. In some cases, at least some of the microglia may make direct contact with dendrites or synapses of the neurons of a subject brain matrix composition. In some embodiments, at least some of the microglia are directly associated with astrocytes of a subject brain matrix composition. In some cases, at least some of the microglia are in direct contact with astrocytes of a subject brain matrix composition. In some embodiments, at least some of the microglia are directly associated with the three- dimensional endothelial vascular network of a subject brain matrix composition. Suitable microglia for use in a subject brain matrix composition may be derived or differentiated from a precursor cell. In some embodiments, the microglia are differentiated from induced pluripotent stem cells (iPSCs). FIG. 3 depicts an exemplary embodiment of differentiation of induced pluripotent stem cells to microglia. Microglia may be selected with flow cytometric sorting for microglial markers (e.g., CD45, CD43, P2RY12).Methods for differentiating iPSCs into microglia are described, for example, in McQuade, Amanda, et al. "Development and validation of a simplified method to generate human microglia from pluripotent stem cells." Molecular neurodegeneration 13.1 (2018): 1-13, the entirety of which is incorporated herein by reference.Oligodendrocytes

[0041] In some embodiments, a subject brain matrix composition may further comprise oligodendrocytes. Oligodendrocytes are myelinating cells of the central nervous system. In some cases, the oligodendrocytes are external to the lumens of a subject three- dimensional endothelial vascular network (i.e., the oligodendrocytes are present on the abluminal side of the three-dimensional endothelial vascular network). In some embodiments, at least some of the oligodendrocytes are directly associated with the neurons of a subject brain matrix composition. In some cases, at least some of the oligodendrocytes are in direct contact with axons of the neurons of a subject brain matrix composition. In some cases, at least some of the oligodendrocytes may form myelin sheaths around axons of the neurons of a subject brain matrix composition. In some embodiments, at least some of the oligodendrocytes are directly associated with astrocytes of a subject brain matrix composition. Suitable oligodendrocytes for use in a subject brain matrix composition may be oligodendrocytes isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the oligodendrocytes are isolated from central nervous system tissue. Methods for isolation and culture of oligodendrocytes are described, for example, in Victoria Sanchez-Gomez, Maria, et al. "Isolation, expansion, and maturation of oligodendrocyte lineage cells obtained from rat neonatal brain and optic nerve." Myelin: Methods and Protocols (2018): 95-113, the entirety of which is incorporated herein by reference. In some embodiments, the oligodendrocytes are differentiated from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Methods for differentiating iPSCs or ESCs into oligodendrocytes are described, for example, in Douvaras, Panagiotis, et al. "Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells." Stem Cell Reports 3, 250-259 (2014), the entirety of which is incorporated herein by reference.Aging and Disease Model Cells

[0042] Suitable cells for use in a subject brain matrix composition include cells that model or recapitulate phenotypes of aging and / or disease states (e.g., neurodegenerative disease states). Any cell type of the present application (e.g., endothelial cells, neurons, pericytes, astrocytes, microglia, oligodendrocytes) may be suitable for modeling or recapitulating phenotypes of aging or disease states. A cell that recapitulates aging phenotypes may, for example, be a primary cell isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the primary cell or precursor cell may be derived from an aging human subject or a human subject with an age-related neurodegenerative disease (e.g., Late Onset Alzheimer's disease or Parkinson’s disease). In some embodiments, the primary cell or precursor cell may be derived from an aging non-human subject or a non-human subject with an age-related neurodegenerative disease (e.g., Late Onset Alzheimer's disease or Parkinson’s disease). In some embodiments, the primary cell or precursor cell may be derived from a cell comprising an allele, mutation, gene deletion, or any other genetic variant that is linked to an accelerated aging or early aging phenotype. In some embodiments, the primary cell or precursor cell may be derived from an animal model of aging or an animal model of an age-related neurodegenerative disease. In some cases, the animal model of aging may be an animal model comprising an allele, mutation, gene deletion, or other genetic variant that is linked to an accelerated aging phenotype. Examples of genetic variants that are linked to accelerated aging include genetic variants associated with Hutchinson-Gilford progeria syndrome (HGPS). Examples of genetic variants that are associated with HGPS include, without limitation, LMNA variants that result in truncated lamin A proteins (e.g., 1824OT), and loss-of-function of ZMPSTE24 (e.g., deletion of all or a portion of ZMPSTE24). Additional examples of genetic variants that are linked to accelerated aging include genetic variants associated with Werner’s syndrome (WS). Examples of genetic variants that are associated with WS include, without limitation, loss-of-function variants of WRN (e.g., R1305X premature stop, R368X premature stop) or deletion of all or a portion of WRN. Other genetic variants that are linked to accelerated aging include genetic variants associated with Wolfram syndrome. Examples of genetic variants that are associated with Wolfram syndrome include, without limitation, loss-of-function variants of WFS1 (e.g., E846K) or deletion of all or a portion of WFS1. Other examples of geneticvariants that are linked to accelerated aging or early aging include genetic variants associated with mitochondrial dysfunction. Genetic variants that lead to mitochondrial dysfunction associated early aging include mutations in POLG that disrupt proof-reading activity of the encoded mitochondrial polymerase (e.g., substitutions of DI 98 and E200). Additional examples of genetic variants that are linked to accelerated aging or early aging include genetic variants associated with chronic inflammation. Examples of genetic variants associated with inflammatory related early aging include, without limitation, loss-of-function variants of NFKB1, IKKP, and IL-10. Further examples of genetic variants associated with accelerated aging or early aging phenotypes include, without limitation, loss-of-function variants and / or deletions of KL (i.e., Klotho), XPD, ERCC1, and TERC. In some embodiments, an accelerated or early aging phenotype may be induced in a subject cell (e.g., a cell derived from an iPSC) by overexpressing a heterologous progerin (i.e., a truncated form of the lamin A protein associated with Hutchinson-Gilford progeria syndrome) or by inhibiting the function of telomerase reverse transcriptase (TERT) to shorten telomere length (e.g., by administration of the TERT inhibitor BIBRI 532). In some embodiments, a subject cell may model or recapitulate an Alzheimer's disease (AD) phenotype. Any cell type of the present application (e.g., endothelial cells, neurons, pericytes, astrocytes, microglia, oligodendrocytes) may be suitable for modeling or recapitulating an Alzheimer's disease (AD) phenotype. A cell that recapitulates an AD phenotype may, for example, be a primary cell isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the primary cell or precursor cell may be derived from a human subject with AD (e.g., late onset or sporadic AD). In some embodiments, the primary cell or precursor cell may be derived from an animal model of AD. For example, the subject neuron may be differentiated from an iPSC derived from a subject with AD. In some embodiments, a subject cell may comprise alleles or mutations associated or linked with AD (e.g., early onset familial AD). For example, a subject cell may comprise an AD linked allele of the amyloid precursor protein (APP) gene. AD linked alleles of APP include, without limitation, K670N, M671L, and V717I alleles. As another example, a subject cell may comprise an AD linked allele of the presenilin 1 (PSEN1) gene. AD linked alleles of PSEN1 include, without limitation, A246E, M146V, and AE9 alleles. As another example, a subject cell may comprise an AD linked allele ofthe presenilin 2 (PSEN2) gene. AD linked PSEN2 genes include, without limitation, the N141I allele.

[0043] In some embodiments, a subject cell may model or recapitulate a Parkinson’s disease (PD) phenotype. Any cell type of the present application (e.g., endothelial cells, neurons, pericytes, astrocytes, microglia, oligodendrocytes) may be suitable for modeling or recapitulating a PD phenotype. A cell that recapitulates a PD phenotype may, for example, be a primary cell isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the primary cell or precursor cell may be derived from a human subject with PD (e.g., sporadic or idiopathic PD). In some embodiments, the primary cell or precursor cell may be derived from an animal model of PD. For example, the subject neuron may be differentiated from an iPSC derived from a subject with PD. In some embodiments, a subject cell may comprise alleles or mutations associated or linked with PD. For example, a subject cell may comprise a PD linked allele of the a-synuclein gene (SNCA). PD linked alleles of SNCA include, without limitation, A53T, A30P, E46K, and G51D alleles. As another example, a subject cell may comprise a PD linked allele of the leucine-rich-repeat kinase 2 (LRRK2) gene. PD linked alleles of LRRK2 include, without limitation, G2019S, G2358R, R1628P, and R1441G alleles. As another example, a subject cell may comprise a PD linked allele of the parkin RBR E3 ubiquitin protein ligase (PRKN) gene. PD linked alleles of PRKN include, without limitation, R42P, T240W, and AE4. As another example, a subject cell may comprise a PD linked allele of the PTEN-induced kinase 1 (PINK1) gene. PD linked alleles of PINK1 include, without limitation, G309D, T313M, and L339P. As yet further examples, a subject cell may comprise, without limitation, PD linked alleles of VPS35, DJ-1, PLA2G6, ATP13A2, FBXO7, POLG, DNAJC6, DNAJC13, TMEM230, SYNJ1, VPS13C, and CHCHD2.

[0044] In some embodiments, a subject cell may model or recapitulate an amyotrophic lateral sclerosis (ALS) phenotype. Any cell type of the present application (e.g., endothelial cells, neurons, pericytes, astrocytes, microglia, oligodendrocytes) may be suitable for modeling or recapitulating an ALS phenotype. A cell that recapitulates an ALS phenotype may, for example, be a primary cell isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the primary cell or precursor cell may be derived from a human subject with ALS. In someembodiments, the primary cell or precursor cell may be derived from an animal model of ALS. For example, a subject neuron may be differentiated from an iPSC derived from a subject with ALS. In some embodiments, a subject cell may comprise alleles or mutations associated or linked with ALS. For example, a subject cell may comprise an ALS linked allele of the superoxide dismutase 1 (SOD1) gene. ALS linked alleles of SOD1 include, without limitation, G93A, A4V, G37R, and Cl 11 Y. As another example, a subject cell may comprise an ALS linked allele of the fused in sarcoma (FUS) gene. ALS linked alleles of FUS include, without limitation, R514G, R521C, R521G, R521H, P525L, and R495X (premature stop). As another example, a subject cell may comprise an ALS linked allele of the chromosome 9 open reading frame 72 (C9ORF72) gene. ALS linked alleles of C9ORF72 include, without limitation, alleles with 30 or more GGGGCC hexanucleotide repeats in the 5’ UTR of C9ORF72, e.g., 30 or more, 40 or more, 50 or more, 80 or more, 100 or more, or 200 or more repeats of GGGGCC. As yet another example, a subject cell may comprise an ALS linked allele of the TAR DNA-binding protein 43 (TDP-43) gene. ALS linked alleles of TDP-43 include, without limitation, A315T, G287S, and M337V.

[0045] In some embodiments, a subject cell may model or recapitulate a prion disease phenotype (e.g., Creutzfeldt- Jakob disease). Any cell type of the present application (e.g., endothelial cells, neurons, pericytes, astrocytes, microglia, oligodendrocytes) may be suitable for modeling or recapitulating a prion disease phenotype. A cell that recapitulates a prion disease phenotype may, for example, be primary cells isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the primary cell or precursor cell may be derived from a human subject with a prion disease. In some embodiments, the primary cell or precursor cell may be derived from an animal model of a prion disease. For example, a subject neuron may be differentiated from an iPSC derived from a subject with a prion disease. In some embodiments, the subject cell may comprise alleles or mutations associated or linked with a prion disease. For example, a subject cell may comprise a prion disease linked allele of the PRNP gene. Prion disease linked alleles of PRNP include, without limitation, P102L, D178N, E200K, and V2101.

[0046] In some embodiments, a subject cell may model or recapitulate a trinucleotide repeat disorder. Any cell type of the present application (e.g., endothelial cells, neurons,pericytes, astrocytes, microglia, oligodendrocytes) may be suitable for modeling or recapitulating a trinucleotide repeat disorder phenotype. A cell that recapitulates a trinucleotide repeat disorder phenotype may, for example, be primary cells isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the primary cell or precursor cell may be derived from a human subject with a trinucleotide repeat disorder (e.g., Huntington’s disease). In some embodiments, the primary cell or precursor cell may be derived from an animal model of a trinucleotide repeat disorder. In some embodiments, a subject cell may model or recapitulate a Huntington’s disease (HD) phenotype. For example, a subject neuron may be differentiated from an iPSC derived from a subject with HD. In some embodiments, a subject cell may comprise alleles or mutations associated or linked with HD. For example, a subject cell may comprise a HD linked allele of the huntingtin (HTT) gene.HD linked alleles of HTT may comprise 36 or more CAG trinucleotide repeats in exon 1, e.g., 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 80 or more, or 100 or more CAG repeats. In some embodiments, a subject cell may model or recapitulate a spinocerebellar ataxia (e.g., spinocerebellar ataxia type 3) phenotype. For example, a subject neuron may be differentiated from an iPSC derived from a subject with a spinocerebellar ataxia. In some embodiments, the subject cell may comprise alleles or mutations associated or linked with a spinocerebellar ataxia. As an example, a subject cell may comprise a spinocerebellar ataxia type 3 (SC A3) linked allele of the ATXN3 gene. SCA3 linked alleles of ATXN3 include, without limitation, ATXN3 alleles with 52 or more CAG repeats in the coding region, e.g., 52 or more, 55 or more, 60 or more, 65 or more, 70 or more, or 80 or more CAG repeats. In some cases, a subject cell may model or recapitulate a Fragile X syndrome (FXS) phenotype. For example, a subject neuron may be differentiated from an iPSC derived from a subject with FXS. In some embodiments, a subject cell may comprise alleles or mutations associated or linked with FXS. As an example, a subject cell may comprise a FXS linked allele of the FMRI gene. FXS linked alleles of FMRI include, without limitation, FMRI alleles with 200 or more CGG repeats in the coding region, e.g., 200 or more, 250 or more, 300 or more, 400 or more, or 500 or more CGG repeats.

[0047] In some embodiments, a subject cell may model or recapitulate a frontotemporal dementia phenotype. Any cell type of the present application (e.g., endothelial cells,neurons, pericytes, astrocytes, microglia, oligodendrocytes) may be suitable for modeling or recapitulating a frontotemporal dementia phenotype. A cell that recapitulates a frontotemporal dementia phenotype may, for example, be primary cells isolated from an in vivo source or may be derived or differentiated from a precursor cell. In some embodiments, the primary cell or precursor cell may be derived from a human subject with frontotemporal dementia. In some embodiments, the primary cell or precursor cell may be derived from an animal model of frontotemporal dementia. A cell may include one or more mutations linked with FTD. FTD linked mutations include, without limitation, mutations in C9orf72, Mapt, Gm, Tdp43.T cells. Monocytes, and Blood components

[0048] As discussed above, the lumens of a subject three-dimensional endothelial vascular network of the present disclosure may be filled with a fluid (e.g., an aqueous fluid, a cell growth medium). In some embodiments, a subject fluid comprises peripheral immune cells, e.g., lymphocytes, such as T cells B cells, and NK cells, monocytes, etc. In some cases, the peripheral immune cells, e.g., T cells, are perfused through the lumens of a subject three-dimensional endothelial vascular network. Methods of isolating, differentiating, proliferating, and / or activating T cells are known in the art and described, for example in Fuss, Ivan J. "Purification of T cell populations." Current protocols in immunology 128.1 (2020): e94, and U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7, 172,869;7,232,566; 7, 175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041. Suitable T cells include, without limitation, naive T cells, memory T cells, effector T cells, regulatory T cells (Tregs), CD4+helper T cells, CD8+cytotoxic T cells, y5 T cells, and genetically engineered T cells (e.g., CAR-T cells). In some cases, the fluid comprises monocytes. In some cases, the monocytes are perfused through the lumens of a subject three- dimensional endothelial vascular network. Methods for isolating and culturing monocytes are well known in the art and described, for example, in WO2013118899A1 and W02008084069A1, the disclosures of which are incorporated herein by reference.Suitable monocytes include, without limitation, classical monocytes (i.e., CD14+CD16'), non-classical monocytes (i.e., CD14dimCD16+), and intermediate monocytes (i.e., CD14+CD16+).

[0049] In some embodiments, a subject fluid comprises blood components. In some cases, the blood components are perfused through the lumens of a subject three- dimensional endothelial vascular network Suitable blood components include, without limitation, erythrocytes, platelets (i.e., thrombocytes), neutrophils, eosinophils, basophils, and B cells. Erythrocytes are hemoglobin-rich cells that are primarily responsible for the transport of oxygen through the circulatory system. In mammals, mature erythrocytes lack nuclei. Platelets play key roles in hemostasis and blood clotting. B cells are antibodyproducing lymphocytes that are key to humoral immunity. Methods for isolating platelets and / or B cells are well known in the art and described, for example, in Dagur, Pradeep K., and J. Philip McCoy Jr. "Collection, storage, and preparation of human blood cells." Current protocols in cytometry 73.1 (2015): 5-1, the entirety of which is herein incorporated by reference. Neutrophils are the most abundant granulocytes in circulation and function as a vanguard against bacterial and fungal infections. Eosinophils are granulocytes involved in fighting bacterial, parasitic, and viral infections and immunoregulation. Basophils are large granulocytes that function in mediating inflammatory responses. Methods for isolating granulocytes are well known in the art and described, for example, in “Recommended Standard Method for Isolating Mononuclear Cells” (www.sigmaaldrich.com / US / en / technical-documents / protocol / clinical-testing-and- diagnostics-manufacturing / hematology / recommended-standard-method), the entirety of which is herein incorporated by reference. Materials and kits for isolating granulocytes, such as the EasySep™ Human Pan-Granulocyte Isolation Kit (www.stemcell.com / products / easysep-human-pan-granulocyte-isolation-kit.html), are also readily available.Organisms

[0050] Any cell of the present disclosure (e.g., a cell of a subject brain matrix composition) may be a mammalian cell. A suitable mammalian cell may include, without limitation, a cell derived from an ungulate(e.g., a pig, a cow, a goat, a sheep), a feline (e.g., a cat), a canine (e.g., a dog), a rodent (e.g., a mouse, a rat), a non-human primate (e.g., macaques, orangutan, gorilla, chimpanzee), a human, and the like.Matrix Scaffolds

[0051] A subject brain matrix composition of the present disclosure may comprise a scaffold matrix. Scaffold matrices provide a physiologically compatible, three- dimensional structural support for the cells of the brain matrix composition. A suitable scaffold matrix enables embedded cells to develop physiologically relevant morphologies, cell-cell interactions, self-organized three-dimensional structures, and microenvironments. In some embodiments a subject scaffold matrix of the present disclosure may comprise a gel. A gel of the present disclosure may be formed by crosslinking one or more precursor molecules. The gel precursor molecules may be polymeric or non-polymeric. Crosslinks may be formed by covalent or non-covalent interactions. Examples of non-covalent interactions include ionic bonds or hydrophobic interactions. In some cases, the gel is a hydrogel (i.e., the gel comprises an interstitial fluid comprising water). In some embodiments, a hydrogel of the present disclosure comprises a polymer. In some cases, the polymer may be synthetic. In some cases, the polymer may be naturally occurring. In some embodiments, the polymer comprises a polypeptide. Examples of suitable polypeptides include, without limitation, gelatins, lysozymes, albumins, collagens, silk proteins (e.g., silk fibroin, spidroin), elastin-like polypeptides, fibrin, low molecular weight peptidic gelators (e.g., fluorenylmethoxy carbonyl (Fmoc)- protected peptides), biomimetic-polypeptides (e.g., synthetic coiled-coil motifs), and peptide amphiphiles (i.e., hydrophilic peptide sequences covalently bonded to a hydrophobic aliphatic segment). In some embodiments, the polypeptides comprise fibrin. In some embodiments, the polymer comprises a polysaccharide. Examples of suitable polysaccharides include, without limitation, hyaluronic acid (HA), alginate, chitosan, dextran, cellulose, and natural or synthetic derivatives thereof. In some embodiments, the polymer comprises a synthetic polymer. Examples of suitable synthetic polymers include, without limitation, polyethylene glycol (PEG), polyvinyl alcohol (PVA), and polyacrylic acid (PAA). In some cases, a subject polymer may be functionalized (i.e., modified with a chemical moiety) that may facilitate cross-linking of the polymers or tune the physical properties of the hydrogel. In some cases, a subject polymer may be functionalized with, for example, maleimide, vinyl sulfone, thiol, or photo-sensitive (e.g., an azide group), and methacrylate moi eties. A subject hydrogel of the present disclosure may be tuned to exhibit a specific stiffness (i.e., the hydrogel’s resistance to deformation from amechanical stress). The stiffness of a hydrogel of the present disclosure may be expressed in Pascals (Pa) a unit of pressure (i.e., newtons per square meter). The stiffness of a subject hydrogel may be tuned by the choice of constituent polymers, varying the ratios of the hydrogel components, varying the molecular weight of the polymers, functionalizing the polymers with varying chemical moieties, varying the cross-linking methods, etc. Methods and examples of tuning hydrogel stiffness are well known in the art, and described for example in Luo, Tianyi, et al. "A review on the design of hydrogels with different stiffness and their effects on tissue repair." Frontiers in bioengineering and biotechnology 10 (2022): 817391 and Cao, Huan, et al. "Current hydrogel advances in physicochemical and biological response-driven biomedical application diversity." Signal transduction and targeted therapy 6.1 (2021): 426, the disclosures of which are herein incorporated by reference. In some embodiments, a hydrogel of the present disclosure may have a stiffness in the range of lOOPa to lOOOPa, e.g., in some cases the hydrogel stiffness is in the range from 150Pa to 950Pa, such as from 200Pa to 900Pa, such as from 250Pa to 850Pa, such as from 300Pa to 800Pa, such as from 350Pa to 750Pa, such as from 400Pa to 700Pa, such as from 450Pa to 650Pa, and including from 500Pa to 600Pa.Stability

[0052] A subject brain matrix composition of the present disclosure may be stable for one or more weeks at an average temperature from 35°C to 38°C, an average atmospheric CO2 concentration from 4% to 6%, and / or an average relative humidity from 85% to 95%. In some cases, a subject brain matrix composition of the present disclosure may be stable for one or more weeks at an average temperature from 36°C to 37°C, an average atmospheric CO2 concentration from 4.5% to 5.5%, and / or an average relative humidity from 88% to 92%. In some cases, a subject brain matrix composition of the present disclosure may be stable for one or more weeks at an average temperature of 37°C, an average atmospheric CO2 concentration of 5%, and / or an average relative humidity of 90%. In some embodiments, a subject brain matrix composition of the present disclosure is stable for one week. In some embodiments, a subject brain matrix composition is stable for one or more weeks, e.g., one or more weeks, two or more weeks, three or more weeks, four or more weeks. By “stable” it is meant that, once established, the viability, physiology, and morphology of the cells of the brain matrix composition (e.g., endothelial cells, neurons, pericytes, astrocytes, microglia, oligodendrocytes) do not vary appreciablyin the absence of an external perturbation. For example, the viability, cells of a subject brain matrix composition may maintain cell-type identity (e.g., expression of characteristic surface markers), intracellular homeostasis (e.g., anabolic and catabolic metabolism, intracellular pH), extracellular homeostasis (e.g., extracellular pH), genetic stability, cell-cell interactions (e.g., cell-cell signaling, cell-cell physical contacts) for one or more weeks. Also encompassed by the term “stable” is the maintenance of the integrity and physical characteristics of matrix scaffold for one or more weeks. Stability of the brain matrix composition may be assessed using a variety of methods. For example, changes in extracellular pH may be indicative of accumulating waste products, dying or lysed cells, or contamination (e.g., bacterial contamination). The stability of a brain matrix composition may be assessed by monitoring the extracellular pH of the brain matrix composition. In some cases, the pH may be monitored using a colorimetric pH indicator in the brain matrix composition (e.g., phenol red). As another example, the stability of a brain matrix composition may be assessed by monitoring viability of one or more cells of the brain matrix composition. Viability may be assessed by means of a suitable viability assay. Methods, reagents, and kits for a variety of cell viability assays are known in the art and readily available. Examples of viability assays include, without limitation, ATP-luciferase assays, membrane permeability assays (e.g., trypan blue staining, tetrazolium reduction assays, fluorescent DNA-binding assays), protease activity assays, and cytoplasmic marker-release assays (e.g., lactate dehydrogenase-release assays, protease-release assays). Further details regarding cell viability assays may be found, for example, in Terry, L. Riss, et al. "Cell viability assay." Assay Guidance Manual [Internet], Eli Lilly & Company and the National Center for Advancing Translational Sciences (2016): 1-31, the disclosure of which is incorporated herein by reference. In some cases, a cell viability assay may be performed in the microfluidic device, e.g., within the brain matrix chamber. In some cases, a cell viability assay may be performed on all or a portion of the brain matrix composition that has been harvested from the device.Microfluidic Devices

[0053] In embodiments of the disclosure, devices are configured as microfluidic devices.A microfluidic device of the present disclosure may allow for the containment and growth of a subject brain matrix composition. A microfluidic device of the present disclosuremay allow for perfusion of a subject fluid through the brain matrix composition (e.g., through the lumens of the three-dimensional endothelial cell vascular network). Additionally, a microfluidic device of the present disclosure may further allow for introduction of a perturbation to a subject brain matrix composition. In some cases, a microfluidic device of the present disclosure may allow for assessment (e.g., imaging of) of a subject brain matrix composition before, concurrent with, and / or after perturbation of the subject brain matrix composition.

[0054] Any convenient microfluidic device may be employed. In some instances, the microfluidic device may be one that is commercially available. Examples of commercially available microfluidic devices that may be employed in embodiments of the invention include, but are not limited to: idenTx™ microfluidic devices (aimbiotech) (see e.g., M. A. Winkelman, D. Y. Kim, S. Kakarla, A. Grath, N. Silvia, G. Dai, Interstitial flow enhances the formation, connectivity, and function of 3D brain microvascular networks generated within a microfluidic device. Lab Chip 22, 170-192 (2021)); and the like. In yet other embodiments, custom made microfluidic devices may be employed.

[0055] An exemplary microfluidic device is illustrated schematically in FIG. 4A in a top- down view. A subject microfluidic device of the present disclosure comprises a brain matrix chamber 1 that may contain a brain matrix composition of the present disclosure. The volume of the brain matrix chamber 1 may be defined by its length, width, and height. The length of the brain matrix chamber 1 runs parallel to the longitudinal axis of the chamber (shown as a dashed line in the exemplary embodiment depicted in FIG. 4A). The height and width of the brain matrix chamber 1 are transverse to the longitudinal axis of the chamber. In some cases, the length, width, and height of the brain matrix chamber 1 are the same. In some cases, the length, width, and height of the brain matrix chamber 1 differ. In some embodiments the length of the brain matrix chamber 1 is in the range from 0.8cm to 2.3cm, e.g., in some cases the length is in the range from 0.9cm to 2.2cm, such as from 1.0cm to 2.1cm, such as from 1.0 cm to 2.0 cm, such as from 1.1cm to 1.9cm, such as from 1.2cm to 1.8cm, such as from 1.3cm to 1.7cm, such as from 1.4cm to 1.6cm, such as from 1.45cm to 1.55cm, such as from 1.47cm to 1.53cm, such as from 1.49cm to 1.51cm, and including from 1.495cm to 1.505cm. In some embodiments, the width of the brain matrix chamber 1 is in the ranges from 0.15cm to 0.45cm, e.g., in some cases thewidth is in the range from 0.2cm to 0.4cm, such as from 0.25cm to 0.35cm, such as from 0.27cm to 0.33cm, such as from 0.28cm to 0.32cm, such as from 0.29cm to 0.31cm, and including from 0.295cm to 0.305cm. In some embodiments, the height of the brain matrix chamber 1 is in the range from 0.025cm to 0.075cm, e.g., in some cases, the height is in the range from 0.03cm to 0.07cm, such as from 0.035cm to 0.065cm, such as from 0.04cm to 0.06cm, such as from 0.045cm to 0.055cm, such as from 0.048 cm to 0.052cm, and including from 0.049 cm to 0.051cm. The shape of the brain matrix chamber 1 may be described by the shape of the cross-section transverse to the longitudinal axis of the chamber. The cross-section of the brain matrix chamber 1 may be any suitable shape, e.g., square, rectangular, circular, oval. A subject brain matrix chamber 1 may comprise one or more fluidically coupled ports 2 that enable placement of a subject brain matrix composition into the brain matrix chamber 1. In some embodiments, a subject brain matrix chamber 1 may comprise a first port 2 positioned at one end, along the longitudinal axis, of the brain matrix chamber 1. In some embodiments, a subject brain matrix chamber 1 may comprise a second port 2 positioned opposite, along the longitudinal axis, of the first port 2. In some embodiments a brain matrix chamber port 2 may have a circular cross-section. In some cases, the cross-sectional diameter of a brain matrix chamber port 2 is in the range from 0.15cm to 0.45cm, e.g., in some cases the width is in the range from 0.2cm to 0.4cm, such as from 0.25cm to 0.35cm, such as from 0.26cm to 0.34cm, such as from 0.27cm to 0.33cm, such as from 0.29cm to 0.31cm, and including from 0.295cm to 0.305cm. An exemplary microfluidic device comprising a brain matrix chamber 1 comprising a first and second port 2 is depicted in FIG. 4B.

[0056] A subject microfluidic device may comprise one or more perfusion channels 3. In some embodiments, a subject microfluidic device comprises a first perfusion channel 3 that is adjacent to, and fluidically coupled with, the brain matrix chamber 1. In some embodiments, the first perfusion channel 3 may comprise a central section and one or more outer sections. The longitudinal axis (shown as a dotted line in the exemplary embodiment in FIG. 4A) of the center section of the first perfusion channel 3 runs parallel to the longitudinal axis of the brain matrix chamber 1 (shown as a dashed line in the exemplary embodiment in FIG. 4A). In some cases, the longitudinal axis of the outer sections of the first perfusion channel 3 (shown as a dotted line in the exemplary embodiment in FIG. 4A) may diverge away from the brain matrix chamber 1 to enablebetter external access. In some cases, the longitudinal axis of the outer sections of the first perfusion channel 3 may diverge up to 90°away from the longitudinal axis of the brain matrix chamber 1. In some embodiments, a subject microfluidic device comprises a second perfusion channel 3 that is adjacent to, and fluidically coupled with, the brain matrix chamber 1 and positioned opposite the first perfusion channel 3. In some embodiments, the second perfusion channel 3 may comprise a central section and one or more outer sections. The longitudinal axis (shown as a dotted line in the exemplary embodiment in FIG. 4A) of the center section of the second perfusion channel 3 runs parallel to the longitudinal axis of the brain matrix chamber 1. In some cases, the longitudinal axis of the outer sections of the second perfusion channel 3 may diverge away from the brain matrix chamber 1 to enable better external access. In some cases, the longitudinal axis of the outer sections of the second perfusion channel 3 may diverge up to 90°away from the longitudinal axis of the brain matrix chamber 1. The volume of a center section of a subject perfusion channel 3 may be defined by its length, width, and height. The length of the center section of a perfusion channel 3 runs parallel to the longitudinal axis of the center section of the channel (shown as a dotted line in the exemplary embodiment depicted in FIG. 4A). The height and width of the center section of a perfusion channel 3 are transverse to the longitudinal axis of the center section of the channel. In some embodiments, the length of the center section of a perfusion channel 3 is in the range from 0.8cm to 2.3cm, e.g., in some cases the length is in the range from 0.9cm to 2.2cm, such as from 1cm to 2.1cm, such as from 1cm to 2cm, such as from 1.1cm to 1.9cm, such as from 1.2cm to 1.8cm, such as from 1.3cm to 1.7cm, such as from 1.4cm to 1.6cm, such as from 1.45cm to 1.55cm, such as from 1.47cm to 1.53cm, such as from 1.49cm to 1.51cm, and including from 1.495cm to 1.505cm. In some embodiments, the width of a perfusion channel 3 is in the range from 0.15cm to 0.45cm, e.g., in some cases the width is in the range from 0.2cm to 0.4cm, such as from 0.25cm to 0.35cm, such as from 0.27cm to 0.33cm, such as from 0.28cm to 0.32cm, such as from 0.29cm to 0.31cm, and including from 0.295cm to 0.305cm. In some embodiments, the height of a perfusion channel 3 is in the range from 0.025cm to 0.075cm, e.g., in some cases, the height is in the range from 0.03cm to 0.07cm, such as from 0.035cm to 0.065cm, such as from 0.04cm to 0.06cm, such as from 0.045cm to 0.055cm, such as from 0.048 cm to 0.052cm, and including from 0.049 cm to 0.051cm. The shape of the center section of aperfusion channel 3 may be described by the shape of the cross-section transverse to the longitudinal axis of the channel. The cross-section of a perfusion channel 3 may be any suitable shape, e.g., square, rectangular, circular, oval. In some embodiments, a subject perfusion channel 3 may comprise one or more fluidically coupled ports 4 that may enable perfusion of a fluid through the perfusion channel 3 and / or the brain matrix chamber 1. In some cases, a subject perfusion channel 3 may comprise a first port 4 positioned at one end, along the longitudinal axis, of the perfusion channel 3. In some cases, a subject perfusion channel 3 may comprise a second port 4 positioned opposite, along the longitudinal axis, from the first port 4. In some embodiments, a port 4 of a subject perfusion channel 3 may have a circular cross-section. In some cases, the cross- sectional diameter of a perfusion channel port 4 is in the range from 0.15cm to 0.45cm, e.g., in some cases the width is in the range from 0.2cm to 0.4cm, such as from 0.25cm to 0.35cm, such as from 0.27cm to 0.33cm, such as from 0.29cm to 0.31cm, and including from 0.295cm to 0.305cm. An exemplary microfluidic device comprising a first and second perfusion channel 3, each comprising a first and second port 4, is depicted in FIG. 4B.

[0057] A transverse cross-section view of the center of an exemplary microfluidic device is depicted in FIG. 4C. In some embodiments the first and / or second perfusion channel 3 is separated from the brain matrix chamber 1 by a boundary wall 5. The length of a boundary wall 5 runs parallel to the longitudinal axis of the brain matrix chamber 1. The height and width of a boundary wall 5 are transverse to the longitudinal axis of the brain matrix chamber 1. In some embodiments, the height of the boundary wall 5 is less than the height of the perfusion channel 3. In some embodiments, the height of a boundary wall 5 is in the range from 0.02cm to 0.07cm, e.g., in some cases, the height is in the range from 0.025cm to 0.065cm, such as from 0.03cm to 0.06cm, such as from 0.035cm to 0.055cm, such as from 0.04cm to 0.05cm, such as from 0.043cm to 0.047, and including from 0.044cm to 0.046cm. The width of a boundary wall 5 may vary. In some embodiments, the width of the boundary wall 5 is in the range from 0.04cm to 0.1cm, e.g., in some cases the width is in the range of 0.05cm to 0.09cm, such as from 0.055cm to 0.085cm, such as from 0.06cm to 0.08cm, such as from 0.065cm to 0.075cm, such as from 0.068cm to 0.072cm, and including from 0.069cm to 0.071cm. In some embodiments, the length of the boundary wall 5 is in the range from 0.8cm to 2.3cm, e.g.,in some cases the length is in the range from 0.9cm to 2.2cm, such as from 1cm to 2.1cm, such as from 1cm to 2cm, such as from 1.1cm to 1.9cm, such as from 1.2cm to 1.8cm, such as from 1.3cm to 1.7cm, such as from 1.4cm to 1.6cm, such as from 1.45cm to 1.55 cm, such as from 1.47cm to 1.53 cm, such as from 1.49cm to 1.51cm, and including from 1.495cm to 1.505cm.

[0058] FIGS. 4D-4E provide top-down and transverse views, respectively, of a microfluidic device according to another embodiment of the invention. While similar to the embodiment depicted in FIGS. 4A-4C, the embodiment illustrated in 5D-5E includes a ‘wedged wall’ rather than straight wall to separate the middle chamber from the side chambers. In such embodiments, while the angle of the wedge may vary, in some instances the angle ranges 100 to 150°, such as 120 to 145°, e.g., 130 to 135°

[0059] A microfluidic device of the present disclosure may be comprised of any suitable material. In some cases, a microfluidic device of the present disclosure is comprised of a polymer. In some cases, the polymer is a thermoplastic polymer. Suitable thermoplastic polymers include, without limitation, polystyrene, polyether ketone, polyethylene terephthalate, polyvinyl chloride, polymethylmethacrylate, cyclic olefin copolymer, polycarbonate, polyetherimide, and polydimethylsiloxane (PDMS). In some embodiments, the polymer is PDMS. In some embodiments, a microfluidic device may be comprised of one or more suitable materials. In some embodiments, a microfluidic device of the present disclosure may be comprised of a thermoplastic polymer and a second material. The second material may be any suitable material. In some embodiments, the second material is glass. In some cases, the glass is a glass coverslip. Glass coverslips are known in the art and readily available. Methods and materials for manufacturing microfluidic devices are well known in the art and described, for example, in “Introduction about soft lithography and polymer molding for microfluidics (https: / / www.elveflow.com / microfluidic-reviews / soft-lithography- microfabrication / introduction-about-soft-lithography-and-polymer-molding-for- microfluidic / ); Fiorini, Gina S., and Daniel T. Chiu. "Disposable microfluidic devices: fabrication, function, and application." BioTechniques 38.3 (2005): 429-446; US Pat. App. No. 2004 / 0115838; WO 2005 / 030822; and WO 2005 / 084191, the disclosures of which are incorporated herein by reference. In some embodiments, the microfluidic device may be produced by soft lithography. In some cases, a thermoplastic polymer(e.g., PDMS) precursor may be poured onto a positive mold of the microfluidic device (e.g., a positive silicon mold) and the PDMS subsequently cured (e.g., by baking) to produce a negative polymeric mold. The negative polymeric mold may then be removed from the positive mold and any appropriate ports formed (e.g., by removing material with a biopsy punch) in the negative polymeric mold. A glass surface (e.g., a glass coverslip) and the corresponding surface of the polymeric negative mold may then be plasma surface treated. Following plasma surface treatment, the glass surface and the corresponding surface of the negative polymeric mold may be bonded together to form the microfluidic device.SYSTEMS

[0060] The present disclosure provides systems comprising a device, e.g., a microfluidic device, of the present disclosure and a reader. The system may be used to assess the effect of a perturbation introduced to the device. The reader may aid in assessing the brain matrix composition in one or more ways. In some embodiments, the reader comprises an imaging system and enables the assessment of the brain matrix composition through imaging of the brain matrix composition. For example, the imaging system may enable imaging of the brain matrix composition to assess the morphology of one or more cells, cell-cell interactions between one or more cells, expression of a protein of interest in one or more cells, and / or localization of a protein of interest in one or more cells before, concurrent with, and after perturbation of the brain matrix composition. In some cases, the imaging system may be used to introduce a perturbation, e.g., an exposure to light. In some embodiments, the reader comprises an electrical measurement system and enables assessment of the brain matrix composition through measurement of the electrical activity in brain matrix composition. For example, an electrical measurement system may enable measurement of electrical activity of one or more neurons of the brain matrix composition before, concurrent with, and after perturbation of the brain matrix composition. In some embodiments, the reader comprises a force measurement system and enables the assessment of the brain matrix composition through measurement of forces in the brain matrix composition. For example, a force measurement system may enable measurement of the perfusion pressure within the lumens of the three-dimensional endothelial cell vascular network before, concurrent with, and after perturbation of the brain matrixcomposition. In some cases, the reader comprises any combination of an imaging system, an electrical measurement system and / or force measurement system as described herein.

[0061] In some embodiments, the system further comprises an environmental control system. An environmental control system may enable regulation of the environmental conditions surrounding a subject microfluidic device. For example, an environmental control system may enable control of the temperature, humidity, and atmospheric conditions surrounding a subject microfluidic device before, concurrent with, and after perturbation of the brain matrix composition. In some cases, the environmental control system may be used to introduce a perturbation, e.g., a change in temperature. In some embodiments, the system further comprises a fluid control system. A fluid control system may enable regulation of the flow of a fluid through the microfluidic device. For example, a fluid control system may regulate the flow of a fluid through the lumens of a three- dimensional endothelial cell vascular network before, concurrent with, and after perturbation of the brain matrix composition. In some cases, the fluid control system may be used to introduce a perturbation, e.g., a mechanical stress. A system of the present disclosure may comprise any combination of reader, environmental control system, and / or fluid control system as described herein.Imaging Systems

[0062] In some embodiments, an imaging system of the present disclosure comprises a magnification device. A magnification device may be any suitable magnification device. A magnification device may comprise one or more objectives. In some embodiments, an objective may be a refractive (i.e., lens) objective. In some embodiments, an objective may be a reflective (i.e., mirror) objective. Suitable objectives may vary in magnification, numerical aperture, focal length, optical aberration, chromatic aberration and working distance. In some embodiments, an imaging system comprises an image sensor. In certain embodiments, an image sensor may comprise a charge-coupled device (CCD) sensor, a complementary metal oxide semiconductor (CMOS) sensor, and photomultipliers (PMTs). In some embodiments, the imaging system comprises a light source. The light source may be any suitable light source. In some cases, the light source may emit a laser beam in the infrared, near-infrared, visible, and / or ultraviolet light range. In some embodiments, the light source may be a gas laser, solid-state laser, a dye laser, a semiconductor laser (e.g., a diode laser), or a fiber laser. In some cases, the light sourcemay further comprise an optical condenser. The optical condenser may be used to render divergent light beams from the light source into parallel or convergent beams, correct for spherical aberration, and / or correct for chromatic aberration. An optical condenser may be any suitable optical condenser. Examples of suitable optical condensers include, without limitation, chromatic condensers, aplanatic condensers, and compound achromatic condensers. In certain embodiments, the imaging system may comprise one or more filters (e.g., a dichroic filter, an excitation filter, an emission filter). In some embodiments, the light from the light source may be passed through one or more pinholes to reject out-of-focus light (e.g., as in confocal microscopy approaches). In some embodiments, an imaging system of the present disclosure may be configured to capture images of the brain matrix composition. In some cases, an imaging system may be configured to illuminate the brain matrix composition. An exemplary embodiment of an imaging system 6 configured to illuminate and capture images of a brain matrix composition of a subject microfluidic device 7 is depicted in FIG. 5. Directly visible in FIG.5 is an optical condenser of the light source of the exemplary imaging system 6.Environmental Control Systems

[0063] In some embodiments, an environmental control system of the present disclosure comprises a temperature control system. In some embodiments, a temperature control system comprises a thermostat. A thermostat may be any suitable thermostat. In certain embodiments, a thermostat may be a mechanical temperature sensor (e.g., comprise a bimetallic strip) or an electronic temperature sensor (e.g., comprise a thermistor, comprise a thermocouple). In some embodiments, a temperature control system may comprise a heating element. The heating element may be any suitable heating element. In some cases, the heating element is a heat block. In some embodiments, an environmental control system of the present disclosure comprises an atmospheric control system. In some embodiments, an environmental control system may comprise an atmospheric control system. An atmospheric control system may regulate the concentration of a gas in the atmosphere surrounding a subject microfluidic device. In some cases, an atmospheric control system may regulate the concentration of carbon dioxide (CO2). In some embodiments, an atmospheric control system may comprise a CO2 sensor. The CO2 sensor may be any suitable CO2 sensor. In certain embodiments, the CO2 sensor comprises a thermal conductivity sensor or a nondispersive infrared sensor (NDIR). Anatmospheric control system may comprise a CO2 reservoir. In some embodiments, an atmospheric control system may regulate the humidity (i.e., the concentration of water vapor) of the atmosphere surrounding a subject microfluidic device. In some embodiments, an atmospheric control system may comprise a humidity sensor. The humidity sensor may be any suitable humidity sensor. In certain embodiments, the humidity sensor may be a hygrostat or humidistat. An atmospheric control system may comprise a humidifier and / or dehumidifier. A humidifier may be any suitable humidifier. A dehumidifier may be any suitable dehumidifier. An atmospheric control system may comprise a water reservoir. An exemplary embodiment of an environmental control system 8 housing an imaging system 6, a subject microfluidic device 7, and fluid control system 9 is depicted in FIG. 6.Fluid Control Systems

[0064] A fluid control system of the present disclosure may comprise a fluid actuator. The fluid actuator may be any suitable fluid actuator. In certain embodiments, the fluid actuator may be a pneumatic pump, a peristaltic pump, piezo-electric pump, or a syringe pump. In some embodiments, a fluid control system may comprise a flow sensor. The flow sensor may be any suitable flow sensor. In certain embodiments, the flow sensor may be a thermal flow sensor (e.g., a calorimetric flow sensor), a mechanical flow sensor (e.g., a cantilever flow sensor), an acoustic flow sensor (e.g., a surface acoustic wave flow sensor), an optical flow sensor, or an electro-magnetic flow sensor. In some embodiments, a fluid control system may comprise a pressure sensor. A pressure sensor may be any suitable pressure sensor. In certain embodiments, a pressure sensor may be a gauge pressure sensor, a differential pressure sensor, or an absolute pressure sensor. In some embodiments, a fluid control system may include a bubble trap. In some embodiments, the fluid control system is fluidically coupled to one or more perfusion channels of a subject microfluidic device. In some cases, the fluid control system may be fluidically coupled to one or more perfusion channel ports of a subject microfluidic device. In some embodiments, the fluid control system is fluidically coupled with the brain matrix chamber of a subject microfluidic device. A fluid control system may be configured to drive and regulate the flow of a fluid through a subject microfluidic device of the present disclosure. In some cases, a fluid control system may drive and regulate the flow of a fluid through one or more perfusion channels of a subject microfluidic device.In some cases, a fluid control system may drive and regulate the flow of a fluid through the brain matrix chamber of a subject microfluidic device. In some cases, a fluid control system may drive and regulate the flow of a fluid through the brain matrix composition in the brain matrix chamber of a subject microfluidic device. A fluid control system may modulate the total volumetric flow rate of a fluid through the brain matrix composition of a subject microfluidic device. The total volumetric flow rate may be described as the volume of fluid that passes through a given mass of brain matrix composition per unit time, e.g., ml / (g min). In some embodiments, the total volumetric flow rate of a fluid through a brain matrix composition is in the range of about 10ml / (100g min) to about 100ml / (100g min), e.g., ml / (g min). In some embodiments, the fluid has a volumetric flow rate from 10ml / (100g min) to 100ml / (100g min), e.g., in some cases the volumetric flow rate is in the range from 10ml / (100g min) to 90ml / (100g min), such as from 20ml / (100g min) to 80ml / (100g min), such as from 30ml / (100g min) to 70ml / (100g min), such as from 40ml / (100g min) to 60ml / (100g min). In some cases, a fluid control system may modulate perfusion of a fluid through the lumens of a three-dimensional endothelial vascular network of a subject brain matrix composition. In some cases, a fluid control system may modulate the average flow velocity of a fluid in the lumens of a three- dimensional endothelial cell vascular network. In some embodiments, the flow velocity is in the range from about O.lmm / sec to about lOmm / sec, e.g., in some cases the flow velocity is in the range from 0.5mm / sec to 9.5mm / sec, such as from Imm / sec to 9mm / sec, such as from 1.5mm / sec to 8.5mm / sec, from 2mm / sec to 8mm / sec, such as from 2.5mm / sec to 7.5mm / sec, such as from 3mm / sec to 7mm / sec, such as 3.5mm / sec to 6.5mm / sec, such as from 4mm / sec to 6mm / sec, and including from 4.5mm / sec to 5.5mm / sec. In some cases, a fluid control system modulates the pressure of a fluid through the lumens of a three-dimensional endothelial vascular network (i.e., the perfusion pressure) of a subject brain matrix composition. In some embodiments, the fluid has a pressure in the range from about 1mm Hg to about 20mm Hg e.g., in some cases the pressure is in the range from 2mm Hg to 19mm Hg, such as from 3mm Hg to 18mm Hg, such as from 4mm Hg to 17mm Hg, such as from 5 mm Hg to 16mm Hg, such as from 6mm Hg to 15mm Hg, such as from 7mm Hg to 14mm Hg, such as from 8mm Hg to 13 mm Hg, such as from 9mm Hg to 12mm Hg, and including from 10mm Hg to 11mm Hg. In some cases, the pressure of the fluid may drive the flow of the fluid through thelumens of a subject three-dimensional endothelial cell vascular network. In some cases, the fluid is aqueous. In some cases, the fluid is a cell growth medium (e.g., endothelial growth medium, Dulbecco’s modified eagle medium, human plasma-like medium). In certain embodiments, the fluid may comprise T cells, monocytes, and / or blood components of the present disclosure. A fluid may have any combination of the attributes discussed above. An exemplary embodiment of a component of a fluid control system 9 fluidically coupled to a subject microfluidic device 7 is depicted in FIG. 6. Directly visible in FIG. 6 is a flexible fluid channel that fluidically couples the subject microfluidic device 7 to a fluid actuator of the subject fluid control system 9.METHODS

[0065] The present disclosure provides methods for preparing a subject device. Aspects of the methods include methods for preparing a subject brain matrix composition within the brain matrix chamber of a subject device, e.g., microfluidic device. Other aspects of the methods include preparing a subject device, e.g., microfluidic device for use in an assay. Methods of preparing a device for use in an assay include introducing T cells, monocytes, and / or blood components into the lumens of a three-dimensional endothelial vascular network of a subject brain matrix composition. Additional aspects of the methods include introducing a perturbation to a subject device. A perturbation may encompass an agent (e.g., a small molecule, a nucleic acid, a peptide, a nanoparticle, a viral vector, a non-viral vector, a virus, a cell) or a change in condition (e.g., a mechanical stress, and exposure to light, a change in temperature, a change in pH). Methods of introducing a perturbation to a device include assessing a subject brain matrix composition before, concurrent with, and / or after introduction of the perturbation.Methods of Preparing a Device

[0066] Methods for preparing a subject device include methods for preparing a brain matrix composition of the present disclosure within the brain matrix chamber of a subject device. Methods for obtaining, producing, and / or culturing individual cell-types of a subject brain matrix composition are discussed above. In some embodiments, the methods of preparing a subject brain matrix composition include centrifuging individual cell-type cultures (e.g., endothelial cells, neurons, astrocytes, microglia, pericytes, oligodendrocytes) to pellet the individual cell types. Pelleted cell types may then beresuspended to a suitable concentration. In some embodiments, subject cells may be resuspended to a concentration in the range of IxlO5cells / ml to IxlO8cells / ml, e.g., in some cases the subject cells may be resuspended to a concentration in the range of 5xl05cells / ml to 5xl07cells / ml, such as from IxlO6cells / ml to IxlO7, such as from 2.5xl06cells / ml to 7.5xl06cells / ml cells / ml, and including from 4xl05cells / ml to 6xl06cells / ml. Pelleted individual cell types may be re-suspended in any suitable fluid. In some cases, the fluid is a cell growth medium (e.g., an endothelial growth medium). In some cases, the growth medium may comprise vascular endothelial growth factor (VEGF). In some embodiments, the VEGF concentration is in the range from Ing / ml to lOOng / ml, e.g., in some cases the VEGF concentration is in the range from 2ng / ml to 9ng / ml, such as from 3ng / ml to 8ng / ml, such as from 4ng / ml to 7ng / ml, and including from 5ng / ml to 6ng / ml. In some embodiments, the resuspension fluid may comprise a scaffold matrix precursor solution (e.g., fibrinogen). In some cases, the cell resuspensions may be kept on ice to slow polymerization of the scaffold matrix precursor. In some embodiments, individual cell-type resuspensions may then be combined to produce a brain matrix composition precursor. In some embodiments, the resuspension fluid may comprise an initiator or catalyst (e.g., thrombin) for polymerization of a subject scaffold matrix precursor. Prior to substantial polymerization of the subject scaffold matrix, the brain matrix composition precursor may be introduced into the brain matrix chamber of a subject microfluidic device. In some cases, the brain matrix composition precursor may be introduced into the brain matrix chamber via a brain matrix chamber port (e.g., by pipetting the brain matrix composition precursor into the subject port). Once introduced, the scaffold matrix may be allowed to polymerize in the brain matrix chamber by incubating at suitable conditions (e.g., at 37°C and 5% CO2) for a length of time (e.g., 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour) suitable for the scaffold matrix to polymerize. In some embodiments, following polymerization of the scaffold matrix, a suitable cell growth medium may be introduced into one or more perfusion channels of the subject microfluidic device. In some cases, the cell growth medium may be introduced into the one or more perfusion channels via one or more perfusion channel ports. In some embodiments, the cell growth medium may be supplemented with a suitable concentration of VEGF to promote growth and vascularization of the subject endothelial cells. Suitable concentrations of supplemental VEGF are in the range from Ing / ml tolOOng / ml, e.g., in some cases the supplemental VEGF concentration is in the range from lOng / ml to 90ng / ml, such as from 20ng / ml to 80ng / ml, such as from 30ng / ml to 70ng / ml, such as from 40ng / ml to 60ng / ml, and including from 45ng / ml to 55ng / ml. A subject device, containing the brain matrix composition precursor within the brain matrix chamber, may then be incubated under suitable conditions (e.g., at 37°C and 5% CO2) for a suitable length of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 5 days or more) to produce a subject brain matrix composition within the brain matrix chamber. Cell growth media may be changed as necessary during the incubation period. In some embodiments, following formation of the brain matrix composition, additional subject endothelial cells may be introduced into one or more perfusion channels of a subject device. In some cases, the additional subject endothelial cells may be introduced into the one or more perfusion channels via one or more perfusion channel ports. The additional subject endothelial cells may be suspended in any suitable cell growth medium (e.g., endothelial growth medium). Following addition of the subject endothelial cells, the microfluidic device may be incubated under suitable conditions (e.g., at 37°C and 5% CO2) for a suitable length of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 5 days or more). Cell growth media may be changed as necessary during the incubation period. In some cases, the additional endothelial cells may form a monolayer of endothelial cells at the interface between the brain matrix composition and surrounding cell growth media in the one or more perfusion channels.

[0067] The present disclosure also provides methods for preparing a device of the present disclosure for use in an assay. The methods comprise introducing cells into the lumens of the three-dimensional vascular networks. The cells introduced into the lumens of the three-dimensional vascular networks may be suspended in a fluid (e.g., an aqueous fluid, a cell growth medium). In some embodiments, the cells are introduced into a perfusion channel that is fluidically coupled to the brain matrix chamber. In some cases, the cells are introduced through a fluid control system that is fluidically coupled to the brain matrix chamber. In some cases, the cells are introduced through a fluid control system that is fluidically coupled to a perfusion channel. In some embodiments, the cells may be introduced into the lumens of the three-dimensional endothelial cell vascular network by the perfusion of a fluid through the lumens. In some embodiments, the cells comprise T cells. Suitable T cells include, without limitation, naive T cells, memory T cells, effectorT cells, regulatory T cells (Tregs), CD4+helper T cells, CD8+cytotoxic T cells, y5 T cells, and genetically engineered T cells (e.g., CAR-T cells). In some embodiments, the cells comprise monocytes. Suitable monocytes include, without limitation, classical monocytes (i.e., CD14+CD16'), non-classical monocytes (i.e., CD14dimCD16+), and intermediate monocytes (i.e., CD14+CD16+). In some embodiments, the methods further comprise introducing blood components into the lumens of the three-dimensional endothelial cell vascular network. The blood components may be suspended in a fluid (e.g., a human plasma-like medium). In some embodiments, the blood components are introduced into a perfusion channel that is fluidically coupled to the brain matrix chamber. In some cases, the blood components are introduced through a fluid control system that is fluidically coupled to the brain matrix chamber. In some cases, the blood components are introduced through a fluid control system that is fluidically coupled to a perfusion channel. In some embodiments, the blood components may be introduced into the lumens of the three-dimensional endothelial cell vascular network by the perfusion of a fluid through the lumens. In some cases, the blood components may further comprise B cells.Perturbations

[0068] The present disclosure also provides methods of introducing a perturbation to a device of the present disclosure. In some embodiments, the perturbation comprises a small molecule. Naturally occurring or synthetic small molecule compounds of interest include numerous chemical classes, such as organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents may include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such molecules may be identified, among other ways, by employing the screening protocols described below. In some cases, the small molecule may be a drug (e.g., rapamycin, erlotinib, cisplatin, lacosamide, ketamine, and the like). In some cases,the small molecule may be a metabolite (e.g., an amino acid, lactic acid, retinol, and the like). In some cases, the small molecule may be a hormone (e.g., a prostaglandin, a steroid, nitric oxide, and the like). In some cases, the small molecule may be a neurotransmitter (e.g., glutamate, gamma-aminobutyric acid, acetylcholine, dopamine, histamine, epinephrine, and the like). In some embodiments, the small molecule may be used in combination with a genetically modified cell of a subject brain matrix composition to modulate expression, translation, or stability of a gene or protein of interest in the cell (e.g., tamoxifen for use with a tamoxifen inducible expression system, Shield- 1 for use with a FKBP degron system). In some cases, the small molecule may be synthetic. In some cases, the small molecule may be derived from an organism (e.g., a bacterium, a fungus, a plant, and the like).

[0069] In some embodiments, the perturbation comprises nucleic acid. In certain embodiments, the nucleic acid is a deoxyribonucleic acid (DNA). Examples of suitable DNAs include, without limitation, double-stranded DNAs, single-stranded DNAs (e.g., an anti-sense oligonucleotide), linear DNA, circular DNA (e.g., a plasmid). In certain embodiments, suitable DNAs may encode one or more polypeptides (e.g., a CRISPR-Cas effector) and / or may encode one or more non-coding RNAs (e.g., a guide RNA). In some cases, the DNA may be an anti-sense oligonucleotide. In some cases, the DNA is an aptamer. Suitable DNAs may comprise DNA regulatory sequences (e.g., promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like) that provide for and / or regulate transcription of an encoded non-coding RNA or polypeptide and / or regulate translation of an encoded polypeptide. In some cases, a suitable promoter is a cell-type specific promoter (e.g., specific to a cell of a subject brain matrix composition of the present disclosure). Cell-type specific promoters are well known in the art, see, e.g., “Tissue-specific Promoters” (https: / / www.invivogen.com / tissue-specific-promoters). In certain embodiments, the nucleic acid is a ribonucleic acid (RNA). Suitable RNAs may include non-coding RNAs (e.g., a guide RNA) or coding RNAs (e.g., coding for a CRISPR-Cas effector). Suitable non-coding RNAs include, without limitation, microRNAs (miRNAs), small interfering RNAs (siRNAs), anti-sense RNAs (asRNAs), guide RNAs (gRNAs). Suitable coding RNAs may comprise translation regulatory elements (e.g., a 5’ methylguanylate cap, 3’ polyadenylation, an internal ribosome entry site, and the like). In some cases, the RNA isan aptamer. A suitable nucleic acid may comprise a nucleic acid modification. In certain embodiments, a nucleic acid modification may be a modified base and / or a backbone modification that may provide the nucleic acid with a new or enhanced feature (e.g., enhanced stability). Examples of suitable nucleic acid modifications include, without limitation, 2’Omethyl modified nucleotides, 2’ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5’ cap (e.g., a 7- methylguanylate cap (m7G)).

[0070] In some embodiments, the perturbation comprises a peptide or polypeptide. The peptide may be a naturally occurring peptide or may be a synthetic peptide. In certain embodiments, the peptide may be a hormone (e.g., angiotensin, vasopressin, insulin, oxytocin, somatostatin, and the like), a cytokine (e.g., chemokines, lymphokines, tumor necrosis factors, inerferons, interleukins, and the like), a growth factor (e.g., vascular endothelial growth factor, transforming growth factors, neurotrophins, neuregulins, and the like ), a neurotransmitter (e.g., endorphins), an antigenic peptide, an antibody, an enzyme and synthetic derivatives thereof. Suitable antibodies may include, without limitation, monoclonal antibodies, single-chain fragment variable antibodies, nanobodies, and the like. In some embodiments, a peptide may be conjugated to a subject small molecule or subject nucleic acid of the present disclosure. The peptide conjugates may facilitate entry of the attached small molecule or nucleic acid into a cell. In some cases, the peptide may be a subject antibody conjugated to a small molecule or nucleic acid. In some cases, the peptide is a receptor / surf ace marker ligand conjugated to a small molecule or nucleic acid. In some cases, the peptide may be a cell-penetrating peptide (also known as a protein transduction domain) conjugated to a small molecule or nucleic acid. The antibody or the ligand of the conjugate may be specific to a receptor or other surface-marker (e.g., a transferrin receptor, a folate receptor, a LDL receptor) present on the surface of a cell of a subject brain-matrix composition (e.g., and endothelial cell). A cell-penetrating peptide may also be fused to any other peptide of the present disclosure. In some embodiments, the peptide may comprise a peptide modification. In certain embodiments, a peptide modification may be a modified side-chain and / or a backbone modification that may provide the nucleic acid with a new or enhanced feature (e.g., enhanced stability). Examples of suitable peptide modifications include, withoutlimitation, D-amino acids, methyl-amino acids, P-amino acids, peptoids, natural amino acid variants (e.g., homoarginine, benzyloxy -tyrosine, P-phenylalanine), and the like.

[0071] In some embodiments, the perturbation comprises a viral vector. A viral vector may encapsulate a subject nucleic acid of the present disclosure. A viral vector may aid in the delivery of a subject nucleic acid to a cell of a subject brain matrix composition. The use of viral vectors for delivery of nucleic acids is well known in the art and described, for example, in Warnock, James N., Claire Daigre, and Mohamed Al-Rubeai. "Introduction to viral vectors." Viral vectors for gene therapy: methods and protocols (2011): 1-25, the entirety of which is incorporated herein by reference. Suitable viral vectors for use in the present methods include, without limitation, adeno-associated virus vectors, adenovirus vectors, and lentivirus vectors.

[0072] In some embodiments, the perturbation comprises a non-viral vector. A non-viral vector may encapsulate or associate with a subject nucleic acid of the present disclosure. A non-viral vector may aid in the delivery of a subject nucleic acid to a cell of a subject brain matrix composition. The use of non-viral vectors for delivery of nucleic acids is well known in the art and described, for example, in Yin, Hao, et al. "Non-viral vectors for gene-based therapy." Nature Reviews Genetics 15.8 (2014): 541-555, the entirety of which is incorporated herein by reference. Suitable non-viral vectors for use in the present methods include, without limitation, lipid-based vectors (e.g., liposomes), and polymeric vectors (e.g., poly(L-lysine)).

[0073] In some embodiments, the perturbation comprises a nanoparticle. A nanoparticle may encapsulate or associate with a subject small molecule, nucleic acid, and / or peptide of the present disclosure. A nanoparticle may aid in the delivery of a subject small molecule, nucleic acid, and / or peptide to a cell of a subject brain matrix composition. The use of nanoparticles for delivery of small molecules, nucleic acids, and / or peptides is well known in the art and described, for example, in Wilczewska, Agnieszka Z., et al. "Nanoparticles as drug delivery systems." Pharmacological reports 64.5 (2012): 1020- 1037, the entirety of which is incorporated herein by reference. Suitable nanoparticles for use in the present methods include, without limitation, lipid-based nanoparticles, polymeric nanoparticles (e.g., polyglycolide), dendrimers, silicate nanoparticles (e.g., mesoporous silica nanoparticles), carbon nanoparticles (e.g., carbon nanotubes), noble metal nanoparticles (e.g. gold nanoparticles), and magnetic nanoparticles.

[0074] In some embodiments, the perturbation comprises a virus. Suitable viruses include viruses that can infect a mammalian cell. A suitable virus may be a single-stranded DNA (ssDNA) virus, a double-stranded DNA (dsDNA) virus, a single-stranded RNA (ssRNA) virus, and a double-stranded RNA (dsRNA) virus. In some cases, the virus is a pathogenic virus. Suitable viruses for use in the present methods include, for example, viruses belonging to the Coronaviridae, Picornaviridae, Astroviridae , Picobirnaviridae, Filoviridae, Retroviridae, Anelloviridae, Herpesviridae, and Adenoviridae families.

[0075] In some embodiments, the perturbation comprises a cell. Cells of interest may include naturally occurring as well as modified cells, e.g., engineered cells, such as modified T-cells or microglia. In certain embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some cases, the prokaryotic cell is a bacterial cell. Suitable bacterial cells for use in the present methods may include gram-negative bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa, Yersinia pestis, Chlamydia trachomatis). In other cases, suitable bacterial cells include gram-positive bacteria (e.g., Streptococcus pneumoniae, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes). In some cases, suitable bacterial cells include pathogenic bacteria (e.g., Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Bacillus anthracis, Shigella sp., Bordetella pertussis, Helicobacter pylori, Brucella abortus, Listeria monocytogenes, Chlamydia trachomatis, Coxiella urnetiid, Mycobacterium tuberculosis, Salmonella enterica, Yersinia pestis, Mycobacterium tuberculosis). In other cases, suitable bacterial cells include bacterial cells from the human microbiome (e.g., from the genus Bifidobacterium, Feacalibacterium, Clostridium, Roseburia, Ruminococcus, Dialister, Lactobacillus, Enterococcus, Staphylococcus, Sphingobacterium, Bacteroides, Tannerella, Parabacteroides, Alistipes, Prevotella, Escherichia, Shigella, Desulfovibrio, Bilophila, Helicobacter, Fusobacterium, Akkermansia). In yet other cases, suitable bacterial cells include model bacteria for use in scientific research (e.g., Escherichia coli, Streptococcus pyogenes, Bacillus subtilis, Caulobacter crescentus). In some embodiments, a eukaryotic cell is a fungal cell. In some cases, the fungal cell is a pathogenic fungal cell. Suitable fungal cells for use in the present methods include fungal cells belonging to the genus Candida, Blastomyces, Histoplasma, Aspergillus, Cryptococcus, Pneumocystis, and Coccidioidomyces . In some embodiments, a eukaryotic cell is a protozoan cell. In some cases, the protozoan cell is apathogenic protozoan cell. Suitable protozoan cells for use in the present methods include protozoan cells belonging to the genus Entamoeba, Giardia, I.eishmania, Balantidum, Plasmodium, and Cryptosporidium.

[0076] In some embodiments, the perturbation comprises a mechanical stress. In some cases, mechanical stress may comprise a modulation in pressure (e.g., an increase in pressure) within the brain matrix composition. The pressure within the brain matrix composition may be modulated by modulating the flow rate and / or pressure of a fluid through the brain matrix composition. In some cases, the mechanical stress may comprise modulating the pressure within the lumens of the three-dimensional endothelial cell matrix. The pressure within the lumens (i.e., perfusion pressure) may be modulated by modulating the flow rate and / or pressure of a fluid in the lumens of the three-dimensional endothelial cell matrix. The flow rate and / or pressure of a fluid in a subject brain matrix composition may, for example, be modulated by a subject fluid control system of the present disclosure. In some cases, the effect or magnitude of a mechanical stress (e.g., a change in pressure of a fluid in a subject brain matrix composition) may be modulated by the given stiffness of the matrix scaffold of a subject brain matrix composition.Analyses

[0077] In some embodiments, the methods of introducing a perturbation to a device of the present disclosure further comprise assessing the brain matrix composition before, concurrent with, and / or after introduction of the perturbation to the microfluidic device. The brain matrix composition may be assessed by any suitable method. In some embodiments, the assessment may comprise observation or measurement of an aspect of the brain matrix composition within the brain matrix chamber. For example, the brain matrix composition may be imaged within the brain matrix chamber. In some embodiments, the assessment may comprise harvesting all or a portion of the brain matrix composition and assessing an aspect of the harvested brain matrix composition. The harvested brain matrix composition may be assessed by any suitable method. For example, the expression of a nucleic acid in the brain matrix composition may be measured.

[0078] In some embodiments, the methods of introducing a perturbation to a device comprise imaging the brain matrix composition. In some cases, the brain matrix composition may be imaged with an imaging system of the present disclosure. In certainembodiments, the brain matrix composition may be imaged before, concurrent with, or after introduction of the perturbation to the device.

[0079] In some embodiments, the methods of introducing a perturbation to a device of the present disclosure further comprises fixing the brain matrix composition. Methods for fixing cells and tissues are well known in the art. The brain matrix composition may be fixed with any appropriate fixative known in the art. Suitable fixatives include, without limitation, formalin, paraformaldehyde, glutaraldehyde, methanol, acetone Bouin’s fixative, Zenker’s solution, Helly’s solution, Camoy’s solution, ethanol, formaldehyde / glutaraldehyde solution, and combinations thereof. In some embodiments, the methods further comprise permeabilizing the brain matrix composition, e.g., to provide access to analytes of interest in a cell of the brain matrix composition. Methods for permeabilizing cells and tissues are well known in the art. Permeabilization may be achieved by heating, and / or by addition of a permeabilizing agent. Suitable permeabilizing agents include, without limitation, saponin, Triton™X-100, di gitonin, Leucoperm™, and Tween®20. In some embodiments, protease treatment can be employed. Suitable proteases include, for example, trypsin, pepsin, and protease type XIV.

[0080] In some embodiments, the methods further comprise immunostaining the brain matrix composition. In some cases, all or a portion of the fixed and / or permeabilized brain matrix composition may be harvested and sectioned prior to immunostaining. In some embodiments, a scalpel may be used to cut away a portion of the brain matrix chamber of the microfluidic device and expose the brain matrix composition. The brain matrix composition may be harvested by any suitable method (e.g., manually with a scalpel and tweezers). The sectioning may be accomplished with any suitable sectioning method for tissues known in the art (e.g., methods for cryotome sectioning of tissues). Methods of immunostaining cells and tissues are well known in the art. Immunostaining may be used to detect an analyte of interest (e.g., a nucleic acid, a protein) in or on a cell of the brain matrix composition. In some cases, a subject brain matrix composition may be contacted with a primary antibody specific to an analyte of interest. In some cases, the brain matrix composition is contacted with a secondary antibody specific to a constant region of the primary antibody. In some embodiments, the primary and / or secondary antibody is conjugated to a fluorophore. Suitable fluorophores include, without limitation,fluorescent proteins (e.g., GFP), fluorescent pigments (e.g., phycoerythrin), fluorescent dyes (e.g., Alexa Fluors). In some embodiments, the primary and / or secondary antibody is conjugated to an enzyme. The enzyme may catalyze a chemiluminescent reaction. Suitable enzymes include, without limitation, hydrolases (e.g., phosphatases, glycosidases), oxidoreductases (e.g., peroxidases, oxidases), and NADH dependent enzymes.

[0081] In some embodiments, all or a portion of the brain matrix composition may be harvested without prior fixation. In some embodiments, a scalpel may be used to cut away a portion of the brain matrix chamber of the microfluidic device and expose the brain matrix composition. All or a portion of the brain matrix composition may be harvested using any suitable method. In some cases, a scalpel may be used to cut the brain matrix composition into two or more portions. All or a portion of the brain matrix composition may be harvested by means of suction (e.g., by means of a biopsy punch). In yet other embodiments, the brain-matrix gel can be dissolved directly on the chip and the cells can then be harvested. Following harvest, the brain matrix composition may be dissociated. A brain matrix composition may be dissociated by any suitable means. In some cases, a brain matrix composition may be dissociated by placing the brain matrix composition in a suitable dissociation solution. Suitable dissociation solutions include tissue-dissociation solutions well known in the art. In some cases, a dissociation solution may comprise proteinases. Suitable proteinases include, without limitation, papain, collagenases and metalloproteinases. In some embodiments, dissociation of the brain matrix composition in the dissociation solution may be aided by periodic gentle mixing. In some cases the dissociation reaction is carried out on ice. The dissociation reaction may be carried out for any suitable period of time (e.g., 1 min, 10 min, 30 min). In some cases, a cell may be isolated from the dissociation solution by any suitable means. In some cases, a cell may be isolated by means of a cell strainer. In some embodiments, two or more cells of a dissociated brain matrix composition may be placed or sorted into different compartments (e.g., a well, an emulsion droplet, a microfluidic droplet) that may facilitate downstream single-cell analyses (e.g., single-cell RNA-seq).

[0082] In some embodiments, the methods further comprise lysing a cell of the brain matrix composition. A cell of the brain matrix composition may be lysed by any suitable method. Methods for lysing cells are well known in the art. In some cases, a cell is lysedby placing the cell in a suitable lysis buffer (e.g., a hypotonic solution), optionally comprising a suitable detergent (e.g., Tween®20, Triton™X-100, NP-40, and / or CHAPs), salts (NaCl, MgCh), buffers (e.g., Tris-EDTA, HEPES), and combinations thereof. Other suitable lysis methods include thermolysis or freeze / thaw lysis.

[0083] In some embodiments, the methods may further comprise isolating a nucleic acid from a lysed cell of the brain matrix composition (e.g., an RNA or DNA). Methods, reagents and kits for isolating nucleic acids are well known in the art. Methods for nucleic acid isolation include, without limitation, organic extraction and precipitation methods, silica column-based methods, and bead-based methods. In some embodiments, the methods further comprise sequencing the nucleic acid. Methods, reagents, and kits for nucleic acid sequencing are well known in the art. In certain embodiments, nucleic acid sequencing methods include Sanger sequencing and next-generation sequencing (e.g., paired-end sequencing, ion-proton sequencing, pyrosequencing, nanopore sequencing). In some embodiments, a nucleic acid may be specifically enriched by hybridization or amplification-based methods. In some embodiments, a nucleic acid may be sequenced by an epigenetic sequencing method (e.g., bisulfite sequencing, ATAC-seq, ChlP-seq). In some embodiments, a nucleic acid may be sequenced by a single-cell sequencing method (e.g., single-cell RNA-seq). Methods, reagents, and kits for single-cell sequencing are known in the art. Suitable single-cell sequencing approaches include, for example, SMARTer, Smart-seq2, MATQ-seq, MARS-seq, CEL-seq, Drop-seq, InDrop, Chromium, SEQ-well, and SPLIT-seq approaches. In some embodiments, the methods may further comprise measuring the expression level of the nucleic acid. In some cases, the method may comprise measuring the expression level of the nucleic acid in the brain matrix composition. In some cases, the method may comprise measuring the expression level of the nucleic acid in a cell type of the brain matrix composition. In some cases, the method may comprise measuring the expression level of the nucleic acid in a single cell of the brain matrix composition.

[0084] In some embodiments, the methods may further comprise isolating a protein from a lysed cell of the brain matrix composition. Methods, reagents, and kits for protein isolation and / or purification are known in the art. Suitable methods for protein isolation and purification may include gel electrophoresis (e.g., 2-dimensional gel electrophoresis) and liquid chromatography (e.g., high performance liquid chromatography). In somecases, methods for protein isolation may optionally include digestion, reduction, alkylation steps, dialysis and / or desalting steps. In some cases, protein isolation may optionally comprise enrichment or depletion steps. Enrichment for a protein of interest may, for example, be accomplished by immunoprecipitation. In some cases, protein isolation may comprise enrichment for a post-translational modification (e.g., phosphorylation, glycosylation). Enrichment of a desired post-translational modification may be accomplished using affinity chromatography employing a suitable affinity ligand (e.g., lectins in the case of glycosylation). In some embodiments, the methods further comprise identifying the protein. In some embodiments, the methods may further comprise identifying a post-translational modification of the protein (e.g., phosphorylation, glycosylation, acetylation). A protein, and / or a post-translational modification (PTM) of the protein, may be identified by any suitable method. Methods, reagents, and kits for protein and / or PTM identification are well known in the art. In some embodiments, the protein may be identified by immunostaining (e.g., a western blot). In some embodiments, the protein may be identified by mass spectrometry. Suitable mass spectrometry approaches for the present methods include matrix assisted laser desorption / ionization mass spectrometry (MALDI-MS), liquid chromatography mass spectrometry (LC-MS) and tandem liquid chromatography mass spectrometry (LC- MS / MS). In some embodiments, the methods may optionally include relative or absolute quantitation methods. Quantitation methods may include stable isotope labeling using amino acids in cell culture (SILAC) and tandem mass tagging (TMT). In some embodiments, the methods may further comprise measuring the expression level of the protein in a brain matrix composition.

[0085] In some embodiments, the methods may further comprise isolating a metabolite from a lysed cell of the brain matrix composition. Methods, reagents, and kits for metabolite isolation and / or purification are known in the art and described, for example, in Clish, Clary B. "Metabolomics: an emerging but powerful tool for precision medicine." Molecular Case Studies 1.1 (2015): a000588, the disclosure of which is incorporated herein by reference. Metabolites may be isolated by methanol and / or acetonitrile extraction followed by hydrophilic interaction chromatography (HILIC), reverse-phase chromatography, or ion-exchange chromatography depending on the physical and chemical properties of the metabolite of interest. In some embodiments, a metabolite maybe identified by mass spectrometry (e.g., LC-MS). In some embodiments, the methods may further comprise quantifying the abundance of the metabolite in a brain matrix composition.

[0086] In some embodiments, the methods may further comprise isolating a lipid from a lysed cell of the brain matrix composition. Methods, reagents, and kits for lipid isolation and / or purification are known in the art. As an example, lipids may be isolated by isopropyl alcohol extraction and reverse-phase chromatography. In some embodiments, a lipid may be identified by mass spectrometry (e.g., LC-MS). In some embodiments, the methods may further comprise quantifying the abundance of the lipid in a brain matrix composition.KITS

[0087] The present disclosure provides kits comprising a device of the present disclosure. The kits may be suitable for practicing embodiments of the methods described herein. In some embodiments, the kit further comprises a population of T cells of the present disclosure. The population of T cells may be present in a suitable container. In some cases, the population of T cells may be cryogenically frozen. In some embodiments, the kit further comprises a population of monocytes of the present disclosure. In some cases, the population of monocytes may be present in a suitable container. In some cases, the population of monocytes may be cryogenically frozen. In some embodiments, the kit further comprises blood components. In some embodiments, the blood components further comprise B cells. The blood components may be present in a suitable container. In some cases, one or more of the individual blood components may be in separate containers. In some one or more of the blood components may be frozen. In some embodiments, the kit further comprises a fresh media of the present disclosure (e.g., endothelial growth medium, DMEM, human plasma-like medium). In some cases, the fresh media may be in a container. A suitable container may be a container that is substantially inert (i.e., does not significantly react with) the cells or blood components contained therein. A suitable container may be resistant to cryogenic freezing temperatures. Examples of suitable materials for the containers include, but are not limited to, glass and plastic. For example, the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREXTM), fused quartz glass, fused silica glass, and the like. Other examples ofsuitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PF A), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like. In some cases, the container may be sealed. The seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an airtight seal that substantially prevents gases from entering or exiting the container. Suitable seals include films or caps. Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethyl ene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof.

[0088] The following examples are offered by way of illustration and not by way of limitation.Examples

[0089] FIG. 6 depicts a schematic of an exemplary microfluidic device of the present disclosure, methods of preparing a microfluidic device for an assay, and methods of introducing a perturbation to a microfluidic device. The brain matrix composition comprises a three-dimensional endothelial cell vascular network. In this example, the microfluidic device may be prepared for use in an assay by introducing T cells, monocytes, and blood components into the lumens of the perfusable three-dimensional endothelial cell vascular network via a perfusion channel of the microfluidic device. The T cells, monocytes, and blood components may perfuse through the three-dimensional endothelial cell vascular network. In this example, a perturbation (e.g., a drug) may be introduced to the microfluidic device via a perfusion channel. The perturbation may perfuse through the three-dimensional endothelial cell vascular network. The effect of the perturbation may subsequently be measured through a variety of analytical techniques (e.g., transcriptomics, metabolomics, electrophysiology, and the like).

[0090] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methodsand structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0091] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0092] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0093] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0094] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0095] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0096] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

WHAT IS CLAIMED IS:

1. An assembly comprising:(a) a brain matrix chamber comprising a brain matrix composition, wherein the brain matrix composition comprises a three-dimensional endothelial cell vascular network and one or more brain parenchymal cells; and(b) a source of a fluid comprising one or more peripheral immune cells fluidically coupled to a lumen of the three-dimensional endothelial cell vascular network.

2. The assembly according to Claim 1, wherein the three-dimensional endothelial cell vascular network comprises brain microvascular endothelial cells.

3. The assembly according to Claim 1, wherein the three-dimensional endothelial cell vascular network comprises human umbilical vein endothelial cells.

4. The assembly according to Claim 3, wherein the human umbilical vein endothelial cells over-express ETV2.

5. The assembly according to any of the preceding claims, wherein the three-dimensional endothelial cell vascular network comprises lumens having an average diameter ranging from 5 pm to 100 pm.

6. The assembly according to any of the preceding claims, wherein brain parenchymal cells comprise microglia.

7. The assembly according to Claim 6, wherein the microglia are external to lumens of the three-dimensional endothelial cell vascular network.

8. The assembly according to any of the preceding claims, wherein the brain parenchymal cells further comprise astrocytes.

9. The assembly according to Claim 8, wherein the astrocytes are external to lumens of the three-dimensional endothelial cell vascular network.

10. The assembly according to any of Claims 8 to 9, wherein a plurality of the astrocytes are directly associated with the three-dimensional endothelial cell vascular network.

11. The assembly according to any of Claims 8 to 10, wherein a plurality of the astrocytes are directly associated with neurons.

12. The assembly according to any of Claims 8 to 11, wherein the astrocytes are derived from fibroblasts.

13. The assembly according to any of Claims 8 to 11, wherein the astrocytes are differentiated from induced pluripotent stem cells.

14. The assembly according to any of the preceding claims, wherein the brain parenchymal cells further comprises neurons.

15. The assembly according to Claim 14, wherein the neurons are external to lumens of the three-dimensional endothelial cell vascular network.

16. The assembly according to any of Claims 14 to 15, wherein a plurality of microglia are directly associated with neurons.

17. The assembly according to any of Claims 14 to 16, wherein a plurality of microglia are directly associated with astrocytes.

18. The assembly according to any of Claims 14 to 17, wherein a plurality of microglia are directly associated with the three-dimensional endothelial cell vascular network.

19. The assembly according to any of Claims 14 to 18, wherein the microglial cells are differentiated from induced pluripotent stem cells.

20. The assembly according to any of the preceding claims, wherein the brain matrix composition further comprises pericytes.

21. The assembly according to Claim 20, wherein the pericytes are external to lumens of the three-dimensional endothelial cell vascular network.

22. The assembly according to any of Claims 20 to 21, wherein a plurality of the pericytes are directly associated with the three-dimensional endothelial cell vascular network.

23. The assembly according to any of Claims 20 to 22, wherein the pericytes are differentiated from induced pluripotent stem cells.

24. The assembly according to any of the preceding claims, wherein the brain matrix composition comprises a gel.

25. The assembly according to Claim 24, wherein the gel is a hydrogel.

26. The assembly according to any of Claims 24 to 25, wherein the gel comprises one or more polymers.

27. The assembly according to Claim 26, wherein one or more of the polymers are proteins.

28. The assembly according to Claim 27, wherein the proteins comprise fibrin.

29. The assembly according to any of Claims 26 to 28, wherein one or more of the polymers are polysaccharides.

30. The assembly according to any of the preceding claims, wherein the assembly is a microfluidic device.

31. The microfluidic device according to Claim 30, comprising a first perfusion channel fluidically coupling the source of the fluid comprising the peripheral immune cells to the lumen of the three-dimensional endothelial cell vascular network.

32. The microfluidic device according to Claim 31, further comprising a second perfusion channel fluidically coupled to a lumen of the three-dimensional endothelial cell vascular network.

33. The microfluidic device according to Claim 32, wherein the first and second perfusion channels are separated from the brain matrix chamber by a boundary structure, where in some instances the boundary structure comprises a wall or pillar.

34. The microfluidic device according to Claim 33, wherein the boundary structure is a wall having a height that is less than the height of the perfusion channel.

35. The assembly according to any of the preceding claims, wherein the lumens of the three- dimensional endothelial cell vascular network are filled with fluid.

36. The assembly according to Claim 35, wherein the fluid comprises peripheral immune cells.

37. The assembly according to Claim 36, wherein the peripheral immune cells comprise T cells.

38. The assembly according to any of Claims 36 to 37, wherein the peripheral immune cells comprise monocytes.

39. The assembly according to any of Claims 36 to 38, wherein the peripheral immune cells comprise B cells.

40. The assembly according to any of Claims 35 to 39, wherein the fluid further comprises blood components.

41. The assembly according to any of the preceding claims, wherein the brain matrix composition is stable for one week or longer.

42. The assembly according to Claim 40, wherein the brain matrix composition is stable for two weeks or longer.

43. The assembly according to Claim 41, wherein the brain matrix composition is stable for three weeks or longer.

44. The assembly according to Claim 42, wherein the brain matrix composition is stable for four weeks or longer.

45. A device comprising:(a) a brain matrix chamber comprising a brain matrix composition, wherein the brain matrix composition comprises a three-dimensional endothelial cell vascular network and one or more brain parenchymal cells; and(b) one or more peripheral immune cells present in a lumen of the three- dimensional endothelial cell vascular network.

46. The device according to Claim 45, wherein the three-dimensional endothelial cell vascular network comprises brain microvascular endothelial cells.

47. The device according to Claim 45, wherein the three-dimensional endothelial cell vascular network comprises human umbilical vein endothelial cells.

48. The device according to Claim 47, wherein the human umbilical vein endothelial cells over-express ETV2.

49. The device according to any of Claims 45 to 48, wherein the three-dimensional endothelial cell vascular network comprises lumens having an average diameter ranging from 5 pm to 100 pm.

50. The device according to any of Claims 45 to 49, wherein brain parenchymal cells comprise microglia.

51. The device according to Claim 50, wherein the microglia are external to lumens of the three-dimensional endothelial cell vascular network.

52. The device according to any of Claims 45 to 51, wherein the brain parenchymal cells further comprise astrocytes.

53. The device according to Claim 52, wherein the astrocytes are external to lumens of the three-dimensional endothelial cell vascular network.

54. The device according to any of Claims 52 to 53, wherein a plurality of the astrocytes are directly associated with the three-dimensional endothelial cell vascular network.

55. The device according to any of Claims 52 to 54, wherein a plurality of the astrocytes are directly associated with neurons.

56. The device according to any of Claims 52 to 55, wherein the astrocytes are derived from fibroblasts.

57. The device according to any of Claims 52 to 56, wherein the astrocytes are differentiated from induced pluripotent stem cells.

58. The device according to any of Claims 45 to 57, wherein the brain parenchymal cells further comprises neurons.

59. The device according to Claim 58, wherein the neurons are external to lumens of the three-dimensional endothelial cell vascular network.

60. The device according to any of Claims 58 to 59, wherein a plurality of microglia are directly associated with neurons.

61. The device according to any of Claims 58 to 60, wherein a plurality of microglia are directly associated with astrocytes.

62. The device according to any of Claims 58 to 61, wherein a plurality of microglia are directly associated with the three-dimensional endothelial cell vascular network.

63. The device according to any of Claims 58 to 62, wherein the microglial cells are differentiated from induced pluripotent stem cells.

64. The device according to any of Claims 45 to 63, wherein the brain matrix composition further comprises pericytes.

65. The device according to Claim 64, wherein the pericytes are external to lumens of the three-dimensional endothelial cell vascular network.

66. The device according to any of Claims 64 to 65, wherein a plurality of the pericytes are directly associated with the three-dimensional endothelial cell vascular network.

67. The device according to any of Claims 64 to 66, wherein the pericytes are differentiated from induced pluripotent stem cells.

68. The device according to any of Claims 45 to 67, wherein the brain matrix composition comprises a gel.

69. The device according to Claim 68, wherein the gel is a hydrogel.

70. The device according to any of Claims 68 to 69, wherein the gel comprises one or more polymers.

71. The device according to Claim 70, wherein one or more of the polymers are proteins.

72. The assembly according to Claim 71, wherein the proteins comprise fibrin.

73. The assembly according to any of Claims 70 to 72, wherein one or more of the polymers are polysaccharides.

74. The device according to any of Claims 45 to 73, wherein the device is a microfluidic device comprising a first perfusion channel fluidically coupling a source of fluid comprising the peripheral immune cells to the lumen of the three-dimensional endothelial cell vascular network.

75. The microfluidic device according to Claim 74, further comprising a second perfusion channel fluidically coupled to a lumen of the three-dimensional endothelial cell vascular network.

76. The microfluidic device according to Claim 75, wherein the first and second perfusion channels are separated from the brain matrix chamber by a boundary structure, where in some instances the boundary structure comprises a wall or pillar.

77. The microfluidic device according to Claim 76, wherein the boundary structure is a wall having a height that is less than the height of the perfusion channel.

78. The device according to any of Claims 45 to 77, wherein the peripheral immune cells comprise T cells.

79. The device according to any of Claims 45 to 78, wherein the peripheral immune cells comprise monocytes.

80. The device according to any of Claims 45 to 79, wherein the peripheral immune cells comprise B cells.

81. The device according to any of Claims 45 to 80, wherein the lumen further comprises blood components.

82. The device according to any of Claims 45 to 81, wherein the brain matrix composition is stable for one week or longer.

83. The device according to Claim 82, wherein the brain matrix composition is stable for two weeks or longer.

84. The device according to Claim 83, wherein the brain matrix composition is stable for three weeks or longer.

85. The device according to Claim 84, wherein the brain matrix composition is stable for four weeks or longer.

86. A system comprising: an assembly according to any of Claims 1 to 44 or a device according to any of Claims 45 to 85; and a reader.

87. The system according to Claim 86, wherein the reader comprises an imaging system.

88. The system according to Claim 87, wherein the imaging system comprises a magnification device.

89. The system according to Claim 88, wherein the imaging system further comprises an image sensor.

90. The system according to any of Claims 86 to 89, wherein the system further comprises a light source.

91. The system according to any of Claims 86 to 90, wherein the imaging system is configured to capture images of the brain matrix composition.

92. The system according to any of Claims 86 to 91, further comprising an environmental control system.

93. The system according to Claim 92, wherein the environmental control system comprises a temperature control system.

94. The system according to any of Claims 92 to 93, wherein the environmental control system further comprises an atmospheric control system.

95. The system according to any of Claims 86 to 94, wherein the system further comprises a fluid actuator.

96. A method of preparing a device for use in an assay, the method comprising introducing peripheral immune cells into the lumens of a three-dimensional endothelial cell vasculature.

97. The method according to Claim 96, wherein the peripheral immune cells comprise T cells.

98. The method according to any of Claims 96 to 97, wherein the peripheral immune cells comprise monocytes.

99. The method according to any of Claims 96 to 98, wherein the method further comprises introducing blood components into the lumens of the three-dimensional endothelial cell vasculature.

100. The method according to any of Claims 96 to 99, wherein the peripheral immune cells comprise B cells.

101. A method comprising introducing a perturbation to an assembly according to any of Claims 1 to 44 or a device according to any of Claims 45 to 85.

102. The method according to Claim 101, wherein the perturbation comprises a small molecule.

103. The method according to Claim 101, wherein the perturbation comprises a nucleic acid.

104. The method according to Claim 101, wherein the perturbation comprises a peptide.

105. The method according to Claim 101, wherein the perturbation comprises a nanoparticle.

106. The method according to Claim 101, wherein the perturbation comprises a viral vector.

107. The method according to Claim 101, wherein the perturbation comprises a non-viral vector.

108. The method according to Claim 101, wherein the perturbation comprises a virus.

109. The method according to Claim 101, wherein the perturbation comprises a cell.

110. The method according to Claim 101, wherein the perturbation comprises a mechanical stress.

111. The method according to Claim 101, wherein the perturbation comprises a change in temperature.

112. The method according to any of Claims 101 to 111, further comprising imaging the brain matrix composition.

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