A model of the blood-brain barrier on a chip

Abstract

Provided is a microfluidic chip for in vitro organoid culture, comprising: a bottom region comprising a center channel and at least one side channel adjacent thereto, the bottom region further comprising a cell-laden medium within the center lane, the cell-laden medium comprising pericytes and astrocytes, and at least one side channel having therein brain microvascular endothelial cells (BMVECs) that form a lumen within the at least one side channel, an upper region; and a porous membrane arranged between the bottom region and the upper region.

Description

A MODEL OF THE BLOOD-BRAIN BARRIER ON A CHIPRELATED APPLICATION

[0001] The present application claims priority to and the benefit of United States patent application no. 63 / 589,155, “A Model Of The Blood-Brain Barrier On A Chip” (filed October 10, 2023). All foregoing applications are incorporated herein by reference in their entireties for any and all purposes.TECHNICAL FIELD

[0002] The present disclosure relates to the field of organ-on-a-chip devices.BACKGROUND

[0003] The blood-brain barrier (BBB), a crucial protection mechanism in the central nervous system (CNS), is a selective barrier of endothelial cells. This barrier, however, hampers the development of CNS therapeutics and diagnostic tools due to the inability of most of these agents to cross the BBB. Thus, BBB models can provide a valuable tool for studying mechanistic aspects related to the transport of drugs at the brain, as well as biological and pathological processes related to the BBB.

[0004] Accordingly, there is a long-felt need for improved BBB models.SUMMARY

[0005] In meeting the described long-felt needs, the present disclosure provides a microfluidic chip for in vitro organoid culture, comprising: a bottom region comprising a center channel and at least one side channel adjacent thereto, the bottom region further comprising a cell-laden medium within the center lane, the cell-laden medium comprising pericytes and astrocytes, and at least one side channel having therein brain microvascular endothelial cells (BMVECs) that form a lumen within the at least one side channel, an upper region; and a porous membrane arranged between the bottom region and the upper region.

[0006] Also provided is a method, comprising: with a device according to the present disclosure, introducing an agent to any one or more of (1) the lumen within the at least one side channel. One can then assess the effect of the agent.

[0007] Further provided is a method, comprising: with a device according to the present disclosure, introducing an agent to any one or more of (1) the lumen within the at least one side channel or (2) the neural progenitor. One can then assess the effect of the agent.

[0008] Additionally provided is a method, comprising: with a device according to the present disclosure, effecting effect a stretching on any one or more of (1) a contents of the at least one side channel and (2) a contents of the center channel. One can then assess the effect of the stretching.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

[0010] FIG. 1 provides a depiction of a device according to the present disclosure. As shown, a central lane (or “gel channel”) having a gel therein can be flanked by one or more vascular channels or “endothelial channels.” PDMS can be used as a substrate on which the lane and channels are defined. As shown, human primary astrocytes and human primary pericytes can be present in the gel of the central lane; BMVECs can be present in the side channel as well as within the gel of the central lane.

[0011] FIG. 2 provides a top view of a chip according to the present disclosure, showing media reservoirs, two endothelial channels, and a gel channel. As shown, endothelial cells and astrocytes can be present in the disclosed system.

[0012] FIG. 3 provides an image showing an example arrangement of CD31 / endothelial cells, GFAP / astrocytes, and DAPI / nuclei in a device according to the present disclosure.

[0013] FIG. 4 provides an illustration of a BBB-on-a-chip system according to the present disclosure that mimics natural tissue. As shown, an arrangement of cells andtissue (right panel) in the disclosed system is similar to a corresponding arrangement in a human cortex.

[0014] FIG. 5 provides an example illustration of the deposition of ECM components in the disclosed technology, showing collagen IV DAPI (left panel) and laminin DAPI (right channel).

[0015] FIGs. 6A-6B provide an example depiction of a device according to the present disclosure that includes neural progenitors. FIGs. 6A-6B relate to a human neurovascular unit (NVU)-on-a-chip model to study TBI. (FIG. 6A) Structure of the NVU. (FIG. 6B) Schematic representation of the NVU-on-a-chip model proposed to understand gender-specific differences following brain injury. As shown, a model can include endothelial cells, pericytes, astrocytes and neurons / neural progenitor cells, which can in turn be termed a neurovascular unit (NVU)-on-a-chip”.

[0016] FIGs. 7A-7B illustrate a 3D network of self-assembled human brain microvasculature in the NVU-on-a-chip. (FIG. 7A). Cell array to be used in the studies and cross-section of an NVU-on-a-chip device (FIG. 7B) Top view of the devices to be used in this study and formation of blood vessels in the main channel. (FIG. 7C)

[0017] FIG. 8 provides an illustration of a BBB-on-a-chip according to the present disclosure with neural progenitors.

[0018] FIG. 9 illustrates an example model for TBI-on-a-chip, as well as example data showing the pressures required to achieve certain degrees of stretching.

[0019] FIGs. 10A-10E illustrate exemplary biological changes in NVU-on-a-chip after uniaxial stretching. (FIG. 10 A) The uniaxial stretching device used in preliminary studies. (FIG. 10B) Decrease in pericyte coverage, (FIG. 10C) Loss of tight junctions, (FIG. 10D) Increase in LDH release, TNF-a secretion, and ROS signaling in TBI-on-a- chip model. (FIG. 10E) Representative images of ROS increase in NVU- and TBI-on-a- chip model.

[0020] FIGs. 11 A-l IB illustrate example isolation and analysis of extracellular vesicles (EVs) from NVU- and TBI-on-a-chip. (FIG. 11 A) The NVU- (control) and TBI- on-a-chip models also indicate a slightly decreased EV concentration following TBI. (FIG. 1 IB) Schematic of example TENPO isolation protocol for neuron- and astrocyte-specific EVs.

[0021] FIGs. 12A-12B provide an example analysis of EV miRNA signatures in NVU- (control) and TBI-on-a-chip models. (FIG. 12 A) miRNA expression in neuron EVs. (FIG. 12B) Changes in the miRNAs in astrocyte EVs.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0022] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0024] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0025] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of' and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as "consisting of' and "consisting essentially of' the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0026] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey thatsimilar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0027] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0028] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0029] As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” can refer to plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9-1.1. Other meanings of “about” can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4. Further, the term “comprising” should be understood as having its open-ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprisescomponents A and B can be a composition that includes A, B, and other components, but can also be a composition made of A and B only. Any documents cited herein are incorporated by reference in their entireties for any and all purposes.

[0030] Any embodiment or aspect provided herein is illustrative only and does not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more embodiments or aspects can be combined with any part or parts of any one or more other embodiments or aspects.

[0031] Traumatic brain injury (TBI), caused by mechanical forces as a result of impact, penetration, or rapid acceleration / deceleration, is a major cause of death and disability worldwide. Depending on its severity, TBI can be classified as mild, moderate, or severe. TBI survivors may experience movement disorders, memory loss, and cognitive deficits, which cause a heavy socio-economic burden. Progressive neurodegenerative proteinopathies such as Parkinson's disease and Alzheimer's disease are the most significant long-term sequelae of TBI. Systematic reviews have found that TBI is associated with a 63%-96% increased risk of all-cause dementia.

[0032] There is also a growing recognition of gender bias in TBI research where fewer women than men are recruited to clinical trials (-30% or less) and male rodents have predominantly been used in injury models. Although TBI prevalence in the general population is 16.7% among males and 8.5% among females, it was found that women are more likely to die from all head injuries (OR = 1.3). One review study performed using 156 published papers comparing clinical outcomes between men and women following TBI revealed that the largest fraction (47%) reported worse outcomes in women than men. What is interesting is that the same study found 44% of the animal studies reported that females fared better than males after TBI. These differences in clinical and animal studies clearly show the need for better preclinical human models to understand biological processes following TBI.

[0033] One of the reasons for these sex differences might be the possible involvement of sex hormones in blood-brain barrier (BBB) formation and neurovascular unit (NVU) development. NVU is a functional unit composed of neurons, astrocytes, and microglia, as well as specialized endothelial cells, mural cells, and the basement membrane (FIG. 6). The blood-brain barrier (BBB) is an active and complex diffusion barrier between the blood and the CNS, which functions as a part of NVU. Studies haveshown that TBI can cause BBB damage and prolonged structural and functional abnormalities of the NVU. Thus, studying gender-specific differences in NVU dysfunction following injury can assist with finding better TBI biomarkers, developing TBI therapies, and understanding TBI-induced neurodegenerative disease pathways.

[0034] Poor understanding of sex differences in TBI pathophysiology and recovery limits clinical care and successful drug development. Given the growing interest in sex as a biological variable affecting injury outcomes and treatment efficacy, there is a clear need to develop gender-specific NVU models. To establish NVU-on-a-chip, endothelial cells, pericytes, and glial cells (astrocytes and microglia) can be embedded in a fibrin hydrogel scaffold to form perfusable, interconnected capillary networks mimicking BBB (FIGs. 7A and 7B). In the NVU, neurons can remain close to the brain capillaries and connect with astrocytic endfeet in near proximity to the BBB. To mimic this structure, neurons can be cultured in the top wells of NVU-on-a-chip devices. Fibrinogen and thrombin obtained from human plasma can be used for fibrin gel formation. Example steps for device preparation can be as follows:

[0035] Step 1 : hBMVECs (e.g., 3 x 106cells / mL), glial cells (e.g., 3 x 106cells / mL), pericytes (e.g., 1 x 106cells / mL) will be suspended in fibrin hydrogel precursor solution (e.g., 10 mg / mL) and mixed with thrombin (e.g., 2U / mL). This mixture will be injected into the main channel of the bottom compartment (labeled in FIG. 7B) to create a cell-laden hydrogel scaffold mimicking NVU.

[0036] Step 2: Neurons and ECs are seeded into the top wells and side channels, respectively. The ECs seeded to the side channels can give rise to an interconnected BBB network. Media introduced into the side channels can maintain the hBMVECs, glial cells, and pericytes. Neuronal medium in the top wells can feed the neurons.

[0037] One can use NVU-on-a-chip devices designed in a “cell array” format, which can contain individual open-top microfluidic chambers, e.g., 15 such chambers (FIG. 7A). This can increase the experimental throughput important for screening different biological responses to be investigated under this project. Culture devices can be prepared, for example, by soft lithography using a silicon elastomer, polydimethylsiloxane (PDMS; Sylgard 184, Dow Coming).

[0038] As a non-limiting example, a microfluidic 3D culture platform composed of hBMVECs, astrocytes, and pericytes were seeded in a physiological ratio (3:3: 1;hBMVEC: Astrocyte: Pericyte) into the middle channel, and neurons on top well (FIG. 10A). Optimization studies showed that fibrin gel can be used as a hydrogel to support all cell populations. hBMVECs embedded in the hydrogel assembled themselves into 3D networks of interconnected endothelial tubes in 5-7 days (FIG. 7B). These engineered brain microvessels also anastomosed with the endothelial lining of the side channels, making it possible to perfuse them when pressure gradient was applied between the two channels.

[0039] In addition, we showed that astrocytes and pericytes closely interacted with these microvessels in our chips. (FIGs. 3-4). The basement membrane, a unique form of the extracellular matrix (ECM) found predominantly under endothelial and epithelial cells, exerts useful functions, such as structural support, cell anchoring, and signaling transduction. In the brain, the most abundant BM proteins are collagen IV, and laminin. Preliminary results also indicated that these BM components are localized especially around the vessels, as seen in vivo.

[0040] After the establishment of the NVU-on-a-chip model, a TBI-on-a-chip model was employed, which model uses uniaxial stretching to mimic concussion in vitro. For these studies, a stretching machine was used, and experiments demonstrated cell death in the NVU model upon stretching (TBI-on-a-chip; FIGs. 10A-10E). Data showed that uniaxial stretching (15% strain) dramatically decreases pericyte coverage (FIGs. 10B and 10C) and tight junction expression (FIG. 10C). In addition, under 15% strain conditions, apoptosis (Caspase 3 / 7 activity) (data not shown), necrosis [lactate dehydrogenase (LDH) release], proinflammatory cytokine (TNF-a) release and oxidative stress [intracellular reactive oxygen species (ROS)] increase compared to control NVU-on-a-chip group (FIGs. 10D and 10E). The changes in the cells upon uniaxial stretching were straindependent.

[0041] Measurement of transcriptomic changes in the TBI-on-a-chip models also advance the molecular understanding of the gender-specific changes following brain injury and contribute to discovering new molecular signatures of TBI. To our knowledge, these types of in-depth studies have not been reported in the literature. Therefore, one can apply single-cell RNA sequencing (scRNA-seq) analysis of micro-physiological systems to investigate whether and how injury alters the transcriptome of engineered human NVU in our model.

[0042] For example scRNA-seq studies, 24 hours after mechanical stretching- induced traumatic brain, cells can be isolated from the devices using an enzyme mixture (trypsin + nattokinase to digest fibrin gel and release the cells), and cell counting is performed with Trypan Blue to detect cell viability. Typically, at least 5,000 cells for each condition will be captured in the lOx genomics single-cell platform to obtain the optimal relationship between the number of cells sampled and the sequencing depth. After sequencing, the data can be imported into Seurat72 in the R environment, processed for QC of low-quality cells and doublets using typical QC metrics (high relative mitochondrial mRNA expression, and barcodes with exceedingly high total transcripts), and then imported into Monocle336 for UMAP projection, clustering, gene expression, and pseudo time trajectory analysis.

[0043] An initial analysis performed using TENPO chips showed that one can isolate EVs from the side channels and top wells of NVU-on-a-chip and TBI-on-a-chip models (FIG. 11). Previous clinical studies showed that EV concentrations (particles / mL) among participants with no TBI were slightly higher than those with a TBI (data not shown). Interestingly, in our model, we saw a similar trend (1.14 x 108 / mL and 1.02 x 108 / mL EVs / mL for NVU- and TBI-on-a-a-chip; FIG. 11 A). We then focused on neuron- and astrocyte-derived EVs and detected a list of EV biomarkers also seen in vivo. Astrocyte EVs in particular showed the enrichment of let7 family members after mechanical stretching mimicking TBI in our system (FIG. 12B).

[0044] Among different BBB models, BBB-on-a-chip can closely recapitulate the key structure and function of the human BBB. A BBB-on-a-chip device according to the present disclosure can be an open-top microfluidic device composed of 2 layers of polydimethyl siloxane (PDMS) slabs separated by a semipermeable membrane. The lower layer of this device has three microchannels separated by two microguides. The middle channel can include a cell-laden gel to form BBB capillaries. To form this gel, human primary microvascular endothelial cells (hBMECs), brain pericytes and astrocytes can be mixed at physiological ratios (3 : 1:3) in a fibrinogen (lOmg / mL) + Matrigel (1 : 1 ratio) precursor solution. Before injecting to the devices, this precursor solution is further mixed with thrombin at concentration of 2U / mL.

[0045] After 24h, 2 side channels are seeded with 3xlO6 / mL endothelial cells. In 7-10 days, BBB capillary structure is formed. For the BBB model containing humanneural progenitor cells, 3xlO6 / mL ReNcell CX or iPS-derived neural progenitor cells are seeded on top of semipermeable membrane separating upper and lower PDMS layers, 24h after seeding of cells to the middle channel. The model also allows for traumatic brain injury simulation. To mimic traumatic brain injury (TBI), we stretch our mature BBB-on- a-chip devices at different values of strain (e.g., 5-15%) in a stretching device. In the model, we demonstrate a 3D BBB model mimicking the natural BBB structure. In this model, capillaries express tight junctions (ZO-1) and deposit ECM components specific to BBB.

[0046] Preliminary Findings

[0047] 1. Establishment of BBB-on-a-chip model with the co-culture of primary human brain microvascular endothelial cells (hBMVECs), primary human pericytes, primary human astrocytes, and human neural progenitor cells:

[0048] We established a 3D BBB model mimicking the natural BBB structure. In this model, capillaries express tight junctions (ZO-1) and deposit ECM components specific to BBB.

[0049] 2. Demonstration of the biological changes upon concussion in a uniaxial stretching in vitro model (TBI-on-a-chip):

[0050] After the establishment of BBB-on-a-chip model, we focused on the development of TBI-on-a-chip model, which uses uniaxial stretching to mimic concussion in vitro. Our data showed that uniaxial stretching (15% strain) dramatically decreases pericyte coverage and tight junction expression. In addition, under 15% strain conditions, oxidative stress [intracellular reactive oxygen species (ROS)], necrosis [lactate dehydrogenase (LDH) release] and apoptosis (Caspase 3 / 7 activity) increased compared to control BBB-on-a-chip group. We also showed that the changes in the cells upon uniaxial stretching are strain-dependent and the increase in strain value increases necrosis, oxidative stress and proinflammatory cytokine (TNF-a) release in BBB-on-a-chip model.

[0051] 3. Determination of biomarkers in TBI-on-a-chip model:

[0052] We performed miRNA analysis for extracellular vesicles (EVs) collected from control and TBI chips. For the initial analysis, we focused on astrocyte -derived EVs and checked the expression of miRNAs detected in animal studies. Our analysis showed that 3 out of 7 markers indicated >1 DCt between TBI and control groups. All these 3 markers had high signal relative to limit of detection (LOD).

[0053] Additional Disclosure

[0054] Traumatic brain injury (TBI), caused by mechanical forces as a result of impact, penetration, or rapid acceleration / decel eration, is a major cause of death and disability worldwide. TBI survivors may experience movement disorders, memory loss, and cognitive deficits, which cause a heavy socio-economic burden. Progressive neurodegenerative proteinopathies such as Parkinson's and Alzheimer's diseases are the most significant long-term sequelae of TBI. Although systematic reviews have found that TBI is associated with a 63%-96% increased risk of all-cause dementia, molecular mechanisms that underlie this risk are mainly unidentified.

[0055] The neurovascular unit (NVU) is a functional unit composed of neurons, astrocytes, microglia, specialized endothelial cells (blood-brain barrier; BBB), mural cells, and the basement membrane. Studies have shown that TBI can cause BBB damage and prolonged structural and functional abnormalities of the NVU. Thus, studying biological processes critical in NVU dysfunction following injury might help find better TBI biomarkers, develop TBI therapies, and understand TBI-induced neurodegenerative disease pathways.

[0056] In this study, we (i) provided an NVU-on-a-chip device that emulates the physiological complexity of the human NVU in ways that have not been possible using conventional techniques and (ii) explored the use of the model to investigate biological responses to the injury and understand underlying molecular mechanisms.

[0057] Materials and Methods

[0058] We use an NVU-on-a-chip model based on the co-culture of brain microvascular endothelial cells (BMVECs), pericytes, astrocytes, and neurons of human origin. As described herein, in this microphysiological system, endothelial cells, pericytes, and astrocytes can be embedded in a fibrin hydrogel scaffold to form perfusable, interconnected capillary networks mimicking BBB. Astrocytes are positioned between neurons and capillary BMVECs and communicate with these cells through their foot processes. To mimic this structure, neurons are cultured in the top wells of NVU-on-a-chip devices. After establishing the NVU-on-a-chip model (used as the “healthy control” in our TBI studies), we focused on developing the TBI-on-a-chip model, which uses uniaxial stretching to mimic concussion in vitro.

[0059] Results, Conclusions, and Discussions

[0060] Our data showed that uniaxial stretching (15% strain) dramatically decreases pericyte coverage and tight junction expression. In addition, under 15% strain conditions, apoptosis and necrosis (Caspase 3 / 7 activity and lactate dehydrogenase (LDH) release), proinflammatory cytokine release, and oxidative stress increase compared to the control NVU-on-a-chip group. We also showed that the changes in the cells upon uniaxial stretching are strain-dependent. Studying transcriptomic differences in the TBI-on-a-chip models by single-cell sequencing can advance the molecular understanding of the changes following brain injury and contribute to discovering new molecular signatures of TBI. Thus, the disclosed 3D, micro-engineered in vitro NVU model allows one to study NVU changes in brain injuries and molecular mechanisms critical in the development of neurodegenerative diseases.

[0061] Aspects

[0062] The following Aspects are illustrative only and do not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more Aspects can be combined with any part or parts of any one or more other Aspects.

[0063] Aspect 1. A microfluidic chip for in vitro organoid culture, comprising: a bottom region comprising a center channel and at least one side channel adjacent thereto, the bottom region further comprising a cell-laden medium within the center lane, the cellladen medium comprising pericytes and astrocytes, and at least one side channel having therein brain microvascular endothelial cells (BMVECs) that form a lumen within the at least one side channel, an upper region; and a porous membrane arranged between the bottom region and the upper region.

[0064] Aspect 2. The microfluidic chip of Aspect 1, further comprising a neural progenitor, the neural progenitor disposed in the upper region and the neural progenitor in fluid communication with the bottom region.

[0065] Aspect 3. The microfluidic chip of any one of Aspects 1-2, further comprising a media reservoir in fluid communication with the lumen of the at least one side channel.

[0066] Aspect 4. The microfluidic chip of any one of Aspects 1-3, wherein the upper region is configured as to permit access to the porous membrane through the upper region.

[0067] Aspect 5. The microfluidic chip of Aspect 4, wherein the upper region comprises an aperture in register with the porous membrane.

[0068] Aspect 6. The microfluidic chip of any one of Aspects 1-5, wherein the bottom region comprises a ridge between the at least one side channel and the center channel.

[0069] Aspect 7. The microfluidic chip of any one of Aspects 1-6, further comprising a mechanism configured to effect a stretching on any one or more of (1) a contents of the at least one side channel and (2) a contents of the center channel.

[0070] Aspect 8. The microfluidic chip of any one of Aspects 1-7, further comprising at least one BMVEC -lined capillary between the cell -laden medium of the center lane and the lumen of the at least one side channel.

[0071] Aspect 9. A method, comprising: with a device according to any one of Aspects 1-7, introducing an agent to any one or more of (1) the lumen within the at least one side channel. One can then assess the effect of the agent.

[0072] Aspect 10. A method, comprising: with a device according to any one of Aspects 2-7, introducing an agent to any one or more of (1) the lumen within the at least one side channel or (2) the neural progenitor. One can then assess the effect of the agent.

[0073] Aspect 11. A method, comprising: with a device according to any one of Aspects 1-7, effecting effect a stretching on any one or more of (1) a contents of the at least one side channel and (2) a contents of the center channel. One can then assess the effect of the stretching. The stretching can be effected by, for example, stretching the bottom region and the top region.

Claims

What is Claimed:

1. A microfluidic chip for in vitro organoid culture, comprising: a bottom region comprising a center channel and at least one side channel adjacent thereto, the bottom region further comprising a cell-laden medium within the center channel, the cell-laden medium comprising pericytes and astrocytes, and at least one side channel having therein brain microvascular endothelial cells (BMVECs) that form a lumen within the at least one side channel, an upper region; and a porous membrane arranged between the bottom region and the upper region.

2. The microfluidic chip of claim 1, further comprising a neural progenitor, the neural progenitor disposed in the upper region and the neural progenitor in fluid communication with the bottom region.

3. The microfluidic chip of any one of claims 1-2, further comprising a media reservoir in fluid communication with the lumen of the at least one side channel.

4. The microfluidic chip of any one of claims 1-2, wherein the upper region is configured as to permit access to the porous membrane through the upper region.

5. The microfluidic chip of claim 4, wherein the upper region comprises an aperture in register with the porous membrane.

6. The microfluidic chip of any one of claims 1-2, wherein the bottom region comprises a ridge between the at least one side channel and the center channel.

7. The microfluidic chip of any one of claims 1-2, further comprising a mechanism configured to effect a stretching on any one or more of (1) a contents of the at least one side channel and (2) a contents of the center channel.

8. The microfluidic chip of any one of claims 1-2, further comprising at least one BMVEC -lined capillary between the cell-laden medium of the center lane and the lumen of the at least one side channel.

9. A method, comprising: with a device according to any one of claims 1-2, introducing an agent to any one or more of (1) the lumen within the at least one side channel.

10. A method, comprising: with a device according to claim 2, introducing an agent to any one or more of (1) the lumen within the at least one side channel or (2) the neural progenitor.

11. A method, comprising: with a device according to claim 1, effecting effect a stretching on any one or more of (1) a contents of the at least one side channel and (2) a contents of the center channel.

Images