Microfluidic chips and biomimetic systems that use them

Abstract

Described herein is a microfluidic chip comprising a first channel in fluid communication with an adjacent second channel through an opening, wherein the heights of the first and second channels are selected to create sufficient surface tension at the opening such that a liquid injected into the first or second channel is substantially confined within the first or second channel, respectively, or such that the flow of the liquid therebetween is controlled, where the surface tension creates a non-physical microfluidic barrier that restricts or selectively controls the passage of the liquid. Also described is an in vitro biomimetic system that uses such microfluidic chips to model the structure and function of human organs, such as the blood-brain barrier, and to study the in vivo-like physiological responses of such organs to various research or therapeutic agents.

Description

[Technical Field]

[0001] Government support clause This invention was made with government support under Contract NIH Training Grant K25 CA201545, allocated by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 013,903, filed on 22 April 2020, the entire disclosure thereof, is incorporated herein by reference in its entirety for all purposes.

[0003] This disclosure relates to an in vitro biomimetic system comprising multiple microfluidic channels for modeling the structure and function of human organs such as the blood-brain barrier and for studying the in vivo-like physiological response of such organs to various studies or therapeutic agents. [Background technology]

[0004] Microfluidic devices, characterized by the diverse structures of multiple microfluidic channels, mimic the structure and function of human biological systems and are used in three-dimensional (3D) cell cultures and organ chip models to investigate the physiological responses of such models to various research or therapeutic agents. Such microfluidic-based human biomimetic systems are highly promising in preclinical drug development because they can provide more accurate physiological responses than conventional cell-based assays and may replace time-consuming and costly in vivo animal testing. Human biomimetic systems utilizing such organ chip models, such as the central nervous system (CNS), blood-brain barrier (BBB), and neurovascular units (NVUs), enable high-throughput, real-time evaluation of organ-specific therapeutic efficacy, toxicity, or disease modeling, effectively reducing the cost of developing new drug and cell therapies. [Overview of the project] [Problems that the invention aims to solve]

[0005] Despite the complexity and cost of in vivo BBB models using animals, microfluidic BBB models present particularly promising applications among many organ-chip models, given that a high percentage of drug candidates that pass animal trials fail in subsequent clinical trials. The BBB provides a homeostatic environment to the CNS and is crucial for healthy brain function. However, the unique barrier properties of the BBB make it difficult to treat CNS disorders because many small and large molecules cannot enter brain regions in amounts sufficient to produce therapeutically meaningful results. Therefore, it is desirable to develop predictive and cost-effective in vitro human BBB models that allow monitoring the delivery efficacy of brain-targeted drugs and investigating pathological neurovascular function in various diseases.

[0006] In recent years, stem cell therapy has emerged as a promising therapeutic treatment for restoring neuronal function after ischemic stroke. However, despite the increasing number of candidate stem cell types being studied, each possessing unique characteristics, there is no effective in vitro assay platform that can systematically evaluate the neurorepairing capacity of candidate cell therapies. When a certain dose of stem cells is transplanted into an ischemic brain, its therapeutic efficacy depends primarily on the response of NVUs to these exogenous cells. While a considerable number of studies support the neurorepairing capacity of stem cells for stroke treatment, some reports contradict some of these observations. This may be partly because the experiments were all conducted under different conditions and / or focused on different aspects of the complex recovery process. Therefore, it would be desirable to develop a consistent and reproducible ischemic stroke model in the form of NVUs on a microfluidic chip, in which cerebral microvascular endothelial cells are directed to form an intact barrier mimicking a functional human blood-brain barrier (BBB), and other constituent cells reproduce in vivo-like behavior in both healthy and ischemic conditions. [Means for solving the problem]

[0007] This disclosure provides microfluidic chips and biomimetic systems using such microfluidic chips, as well as methods for preparing and using them to model, for example, the structure and function of various tissues.

[0008] In some embodiments, the Disclosure provides a microfluidic chip comprising: a plane; a first channel formed on the plane and having a first volume defined by a first width, a first height, and a first length, wherein the first volume extends in a first direction; and a second channel formed on a plane adjacent to the first channel and having a second volume defined by a second width, a second height, and a second length, wherein the second volume extends in a first direction and the second height is greater than the first height, wherein the first channel is the first A first channel is in fluid communication with a second channel through a first opening extending along at least a portion of a length of 1, the first opening extending from a plane to a first height; the first and second heights are sized to generate sufficient surface tension at the first opening such that the liquid injected into the first channel or the second channel is substantially confined within a first volume or a second volume, respectively, or the flow of liquid between them is controlled, the surface tension creating a non-physical microfluidic barrier that restricts or selectively controls the passage of the liquid.

[0009] In some embodiments, such a microfluidic chip further includes a third channel formed on a plane adjacent to a first channel, having a third volume defined by a third width, a third height, and a third length, wherein the third volume extends in a first direction and the third height is greater than the first height, the third channel being in fluid communication with the first channel through a second opening extending along at least a portion of the first length, the second opening extending from the plane to a first height; the first and third heights are sized to generate sufficient surface tension at the second opening such that a liquid injected into the first or third channel is substantially confined within the first or third volume, respectively, or the flow of liquid between them is controlled, the surface tension generating a second non-physical microfluidic barrier that restricts or selectively controls the passage of liquid.

[0010] In some embodiments, such a microfluidic chip further includes a third channel formed on a plane adjacent to a second channel, having a third volume defined by a third width, a third height, and a third length, wherein the third volume extends in a first direction and the third height is lower than the second height, the third channel being in fluid communication with the second channel through a second opening extending along at least a portion of the second length, the second opening extending from the plane to a third height; the second and third heights are sized to generate sufficient surface tension at the second opening so that a liquid injected into the second or third channel is substantially confined within the second or third volume, respectively, or so that the flow of liquid between them is controlled, the surface tension generating a second non-physical microfluidic barrier that restricts or selectively controls the passage of liquid.

[0011] Aspects of the present disclosure further include a biomimetic system comprising a microfluidic chip described herein; and an extracellular matrix confined within a first volume of a first channel, wherein sidewalls of the extracellular matrix extend across a first opening and form a non-physical microfluidic barrier between the first channel and a second channel. <​​​​​​​​​​​​​​​​​​​​​Aspects of this disclosure also include a method for screening a therapeutic agent, the method comprising depositing the therapeutic agent into a second channel of a microfluidic chip of a biomimetic system described herein, and imaging the microfluidic chip.

[0018] Further aspects of the present disclosure include a method for screening a therapeutic agent, the method comprising: carrying out a method for preparing a biomimetic system as described herein; depositing the therapeutic agent into a second channel of a microfluidic chip; and imaging the microfluidic chip.

[0019] In some embodiments, the therapeutic agent comprises stem cells, small molecules, or peptides. A brief explanation of some of the diagrams In the diagram, the same reference number is used to identify similar elements. The size and relative position of elements in the diagram are not necessarily drawn to scale, and some of these elements have been enlarged and positioned to improve the readability of the diagram. Furthermore, the specific shapes of the elements depicted are not intended to convey any information about the actual shape of those elements, but are selected solely for ease of recognition in the diagram. [Brief explanation of the drawing]

[0020] [Figure 1] This is a perspective view of a microfluidic chip according to one embodiment of the present disclosure. [Figure 2] This is a cross-sectional view of a microfluidic chip according to one embodiment of the present disclosure. [Figure 3] This is a top view of a microfluidic chip according to one embodiment of the present disclosure. [Figure 4] This is a top view of a microfluidic chip according to one embodiment of the present disclosure. [Modes for carrying out the invention]

[0021] This disclosure relates to a microfluidic chip comprising a first channel having a first height and a second channel having a second height greater than the first height, wherein the first channel is in fluid communication with the second channel through a first opening, and the first and second heights are sized to generate sufficient surface tension at the first opening such that a liquid injected into the first or second channel is substantially confined within the first or second channel, or the flow of liquid between them is controlled, thereby creating a non-physical microfluidic barrier that restricts or selectively controls the passage of liquid. Also described are biomimetic systems comprising such a microfluidic chip, as well as methods for manufacturing and using the same.

[0022] The microchips and biomimetic systems of this disclosure have one or more of the following advantages:

[0023] (a) There are no physical structures (e.g., capillary pressure barriers or membranes) at the openings between adjacent channels. This not only allows for undisturbed interactions at the interchannel interface but also ensures that the epithelial layer on the sidewalls of the extracellular matrix at the openings between adjacent channels is continuous and intact. Capillary pressure barriers can interfere with cellular interactions at the interchannel interface and can cause physical defects in the epithelial layer. Such defects can lead to shortcuts through the epithelial layer for therapeutic agents. Therefore, a continuous and intact epithelial layer leads to consistent evaluation of therapeutic agents.

[0024] (b) The MPS described above uses patient-derived cells and enables personalized screening models that simulate the pathophysiological state unique to each individual patient.

[0025] (c) The in vitro ischemic stroke models described show induced inflammation and decreased tissue integrity, as well as intrinsic neuroprotection and tissue remodeling.

[0026] Before describing this disclosure in more detail, it would be helpful to provide definitions of certain terms used herein. Additional definitions are provided throughout this disclosure.

[0027] As used herein, the term “channel” refers to a microfluidic channel, i.e., a sealed passage formed on a layer. A channel has a volume defined by width, height, and length, of which at least one is within the sub-millimeter range. To be understood, the term channel encompasses straight channels, as well as channels having portions extending in two or more directions (i.e., channels with bends or curves) and branched channels. A channel typically has an inlet into which a certain volume of liquid can be injected. A channel also optionally has an outlet or vent. The volume enclosed by a microfluidic channel is typically in the microliter or sub-microliter range. In some embodiments, the cross-sectional dimensions of a channel are less than 1 millimeter, less than 500 micrometers, less than 100 micrometers, less than 50 micrometers, or less than 25 micrometers.

[0028] Biomimetic systems, also known as "organ chips," refer to microfabrication platforms designed to model the functional units of organs in vitro. Biomimetic systems enable close contact between different cell types (e.g., between epithelium and vascular endothelium) and simultaneously generate spatiotemporal gradients of chemicals and mechanical strains to mimic organ function.

[0029] As used herein, “living tissue” refers to a collection of functionally interconnected cells cultured and / or assayed using the methods described herein. The cells may be cell aggregates or specific tissue samples from a patient. For example, “living tissue” includes organoids, tissue biopsies, tumor tissue, excised tissue material, and embryos.

[0030] As used herein, the term “stem cell” refers to a totipotent or pluripotent progenitor cell capable of giving rise to various mature human cell lineages. In other words, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells.

[0031] As used herein, the term "small molecule" refers to an organic compound with a size of approximately 1 nanometer (nm) and a low molecular weight (<900 daltons) that may possess some biological activity.

[0032] A "peptide" refers to a polymer of amino acid residues. Peptides include naturally occurring amino acid polymers, non-naturally occurring amino acid polymers, and amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids.

[0033] As used herein, “amino acids” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group (e.g., homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium). Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids.

[0034] A "probe" is a group of atoms or molecules that can be used to detect an analyte. The measurable properties of the probe change in response to the analyte. A "luminescent probe" refers to a probe or molecule that emits light. Types of luminescent probes include bioluminescent, chemiluminescent, electrochemiluminescent, electroluminescent, and photoluminescent. The probe may emit light on its own, or the resulting emission may be the result of a chemical or enzymatic reaction in which the luminescent probe is involved. Alternatively, the probe may be fluorescent.

[0035] "Fluorescence" refers to molecules that can absorb light of a specific frequency and emit light of a different frequency.

[0036] The term "polymer" refers to materials containing macromolecules, which are composed of repeating subunits. Each subunit is called a monomer. Polymers can be natural, semi-synthetic, or synthetic.

[0037] The use of the words "optional" or "optionally" means that the event or situation described may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not occur.

[0038] Where used herein, the term “about” means ±20%, ±10%, ±5%, or ±1% of the indicated range, value, or structure unless otherwise specified. Where used herein, the terms “a” and “an” should be understood to refer to “one or more” of the enumerated components. The use of alternatives (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0039] Unless the context requires otherwise, the word “comprise,” as well as its variations such as “comprises” and “comprising,” and its synonyms and variants such as “include” and “have,” should be interpreted in an open and inclusive sense throughout this specification and the claims. That is, the enumeration of items in the list is “including, but not limited,” so as not to exclude other similar items that may also be useful in the materials, compositions, devices, and methods of the present art. In this specification, the open-ended term “comprising” is used to describe and claim this disclosure as a synonym for terms such as including, containing, or having; however, the present art or its embodiments may instead be described using more restrictive terms such as “consisting of” or “consisting essentially of” the enumerated components.

[0040] Unless otherwise defined, all technical and scientific terms herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0041] Throughout this specification, any reference to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of this disclosure. Therefore, occurrences of the phrase “in one embodiment” or “in another embodiment” in various places throughout this specification do not necessarily all refer to the same embodiment. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, and consequently, any statement that an embodiment may or may include a particular element or feature does not preclude other embodiments of the Art that do not include that element or feature. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0042] In this specification, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the described range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified. Similarly, any number range described herein with respect to any physical characteristics such as polymer subunits, size, or thickness should be understood to include any integer within the described range, unless otherwise specified.

[0043] The following description includes certain specific details to provide a complete understanding of the various embodiments of this disclosure. However, those skilled in the art will understand that this disclosure can be practiced without these details.

[0044] Microfluidic chips As described above, this disclosure provides a microfluidic chip having channels of different heights. A perspective view of one embodiment of the microfluidic chip of this disclosure is shown in Figure 1. A cross-section of the microfluidic chip of Figure 1 is shown in Figure 2.

[0045] The microfluidic chip 100 includes a plane 102 on which a first channel 104 is formed. The first channel 104 has a first volume 106 extending in a first direction. The first volume 106 is defined by a first width 110, a first height 112, and a first length 114. A second channel 116 is formed on the plane 102 adjacent to the first channel 104. The second channel 116 extends in a first direction and has a second volume 118 defined by a second width 120, a second height 122, and a second length 124. As shown, the second height 122 is greater than the first height 112.

[0046] The first channel 104 is in fluid communication with the second channel 116 through a first opening 126 (shown in Figure 2) that extends along at least a portion of the first length 114, the first opening 126 extending from a plane 102 to a first height 112. In various embodiments, the first opening 126 extends along 25% to 75% of the first length 114. In a particular embodiment, the first opening extends along approximately 50% of the first length.

[0047] In the microfluidic chip of this disclosure, the first opening 126 is not obstructed. In other words, there is no physical barrier such as a capillary pressure barrier (e.g., a phase guide, rim, ridge, pillar, etc.) or a membrane at the first opening 126. Instead, the first height 112 and the second height 122 are sized to create sufficient surface tension at the first opening 126 so that the liquid injected into the first channel or the second channel is substantially confined within the first volume or the second volume, respectively, or so that the flow of liquid between them (i.e., between the first volume and the second volume) is controlled, and the surface tension creates a non-physical microfluidic barrier that restricts or selectively controls the passage of liquid. In other words, the difference between the first height and the second height provides sufficient surface tension so that the liquid injected into the first channel does not spread through the opening into the second channel.

[0048] Accordingly, the present disclosure provides a microfluidic chip comprising: a plane; a first channel formed on the plane and having a first volume defined by a first width, a first height, and a first length, wherein the first volume extends in a first direction; and a second channel formed on a plane adjacent to the first channel and having a second volume defined by a second width, a second height, and a second length, wherein the second volume extends in a first direction and the second height is greater than the first height, wherein the first channel has at least a first length A first opening extends along a portion of the first channel to a first height; the first and second heights are sized to generate sufficient surface tension at the first opening so that the liquid injected into the first channel or the second channel is substantially confined within a first volume or a second volume, respectively, or so that the flow of liquid between them (i.e., between the first volume and the second volume) is controlled, thereby creating a non-physical microfluidic barrier that restricts or selectively controls the passage of the liquid.

[0049] Sufficient surface tension is generated when atmospheric pressure is lower than the capillary pressure within the microfluidic channel. As can be understood, the capillary pressure within a microfluidic channel (Pc) can be calculated based on the following formula:

[0050]

number

[0051] Here, γ = surface tension of the liquid in the microfluidic channel h = channel height w = channel width

[0052] θ bottom θ top θ left θ right = The contact angles of the bottom, top, left, and right of the liquid injected into the microfluidic channel.

[0053] The difference in height between adjacent channels allows Pc to be higher than atmospheric pressure Pa, according to the following equation:

[0054]

number

[0055] In embodiments, the microfluidic chip of the present disclosure comprises at least three channels having a first opening between a first channel and a second channel, and a second opening between a first channel and a third channel (discussed in further detail below). In such a configuration, there are no left or right surfaces with respect to the central (i.e., first) channel. Therefore, θ left =θ right = 0.

[0056]

number

[0057] Pa = Atmospheric pressure In various embodiments, the first height 112 is at least 90% of the second height 122. In some embodiments, the first height 112 is at least 80% of the second height 122. In some embodiments, the first height 112 is at least 70% of the second height 122. In some embodiments, the first height 112 is at least 60% of the second height 122. In some embodiments, the first height 112 is at least 50% of the second height 122. In further embodiments, the first height 112 is at least 25% of the second height 122. In certain embodiments, the first height 112 is at least 10% of the second height 122. It is preferable to minimize the difference between the first height and the second height as much as possible in order to maximize the size of the opening and, consequently, the size of the contact area between the channels.

[0058] The specific dimensions of each channel can be modified for specific purposes or designs. In some embodiments, the first height is in the range of 10 micrometers (μm) to 900 μm. In some embodiments, the first height is in the range of 10 μm to 720 μm. In some embodiments, the first height is in the range of 75 micrometers (μm) to 720 μm. In some embodiments, the first height is in the range of 10 μm to 450 μm. In some embodiments, the first height is in the range of 75 μm to 450 μm. In certain embodiments, the first height is approximately 100 μm. In some embodiments, the first width is in the range of 1.25 millimeters (mm) to 2.5 mm. In certain embodiments, the first width is approximately 1.5 mm. In other embodiments, the first width is approximately 2 mm. In some embodiments, the second height is in the range of 300 μm to 1000 μm. In some embodiments, the second height is in the range of 300 μm to 800 μm. In further embodiments, the second height is in the range of 300 μm to 500 μm. In certain embodiments, the second height is approximately 400 μm. In other specific embodiments, the second height is approximately 700 μm. In some embodiments, the second width is in the range of 0.75 mm to 1.5 mm. In certain embodiments, the second width is approximately 1 mm. In certain embodiments, the first height is approximately 100 μm, the first width is approximately 2 mm, the second height is approximately 400 μm, and the second width is approximately 1 mm.

[0059] Various channels can have any suitable length. Since the length of the channel does not affect the surface tension generated at the openings between channels, other factors can be considered when selecting the length of the channel. For example, the length of a particular channel may be selected in part to minimize contamination between channels (e.g., by increasing the distance between the inlets, outlets, or vents of adjacent channels). As shown in Figure 3, the second length 124 may be significantly longer than the first length 114 to space the ends of the channels apart. In various embodiments, the first length is in the range of 5 mm to 10 mm. In a particular embodiment, the first length is about 8 mm. In some embodiments, the second length is in the range of 1.5 cm to 2.5 cm. In a particular embodiment, the second length is about 2 cm. In some embodiments, the length of the second channel is in the range of 0.5 cm to 1.5 cm. In a particular embodiment, the second length is about 1 cm. In a specific embodiment, the first length is about 8 mm and the second length is about 2 cm. In a microfluidic chip having more than two channels, for example three channels, the length of the third channel may be the same as the length of the second channel. In other embodiments, the length of the third channel is longer than the length of the second channel. In yet another embodiment, the length of the third channel is shorter than the length of the second channel. In yet another embodiment, the lengths of the second and / or third channels are shorter than the length of the first channel.

[0060] As described above, each microfluidic channel may be provided with an inlet and an outlet or vent, as required for a particular application. In various embodiments, each microfluidic channel is provided with an inlet and an outlet to facilitate the filling, discharge, and perfusion of liquid. Such inlets and outlets are generally formed as apertures in the cover layer, but other configurations are also conceived.

[0061] As shown in Figures 1 and 2, the microfluidic chip of the present disclosure may include a third channel 128 adjacent to the first channel 104. The third channel 128 extends in a first direction and has a third volume 130 defined by a third width 132, a third height 134, and a third length 136. In the illustrated configuration, the third channel 128 is in fluid communication with the first channel 104 through a second opening 138 extending along at least a portion of the first length 114. The second opening 138 extends from the plane 102 to the first height 112. As shown, the third height 134 is greater than the first height 112. Furthermore, the first height 112 and the third height 134 are sized to generate sufficient surface tension at the second opening 138 so that the liquid injected into the first channel 104 or the third channel 128 is substantially confined within the first volume 106 or the third volume 130, respectively, or so that the flow of liquid between them (i.e., between the first volume and the third volume) is controlled, and the surface tension generates a second non-physical microfluidic barrier that restricts or selectively controls the passage of liquid. The first height 112 and the third height 134 are sized in the same way as described above with respect to the first height 112 and the second height 122. In some embodiments, the second height 122 and the third height 134 are the same.

[0062] In another embodiment, a third channel is formed on a plane adjacent to the second channel. The third channel extends in the first direction and has a third volume defined by a third width, a third height, and a third length. In this configuration, the third channel is in fluid communication with the second channel through a second opening that extends along at least a portion of the third length. The second opening extends from the plane to a third height lower than the second height. Furthermore, the second and third heights are sized to generate sufficient surface tension at the second opening so that the liquid injected into the second or third channel is substantially confined within the second or third volume, respectively, or so that the flow of liquid between them (i.e., between the second and third volumes) is controlled, and the surface tension generates a second non-physical microfluidic barrier that restricts or selectively controls the passage of liquid. Thus, the second and third heights are sized in the same way as described above with respect to the first and second heights. In some embodiments, the first height and the third height are the same.

[0063] Although the microfluidic chips in Figures 1 and 2 are illustrated with three channels, it will be apparent from the above description that the microfluidic chips of this disclosure may have any suitable number of channels. An exemplary two-channel microfluidic chip is shown in Figure 4. As discussed above with respect to the first and second channels, any suitable number (e.g., two, three, four, five, six, etc.) and arrangement of channels can be used, as long as the height difference between adjacent channels provides sufficient surface tension at each opening.

[0064] Furthermore, the microfluidic chips of this disclosure may be in a multi-array format, also known as a multi-well format, to enable use in high-throughput screening formats, such as in vitro cell-based assays, drug screening assays, and toxicity assays. For example, a multi-array culture plate in which 6, 12, 24, 48, 96, 384, or 1536 sample wells (e.g., a multi-channel arrangement for testing a single sample) are arranged in a rectangular matrix. In embodiments, the microfluidic chips conform to one or more dimensions of the standard ANSI / SLAS microtiter plate format.

[0065] The microfluidic chips of this disclosure can be constructed using any suitable technique, such as photolithography, hot embossing, soft embossing, etching, replication molding, or injection molding.

[0066] Biomimetic systems and methods for fabricating them Biomimetic systems (MPS) comprising the microfluidic chip of the present disclosure are also described herein. Such an MPS includes an extracellular matrix confined within a first volume of a first channel. The sidewalls of the extracellular matrix extend across the first opening, forming a non-physical microfluidic barrier between the first channel and the second channel.

[0067] The extracellular matrix can be any suitable gel on which epithelial cells can be cultured. For example, the extracellular matrix may include synthetic or natural polymers (e.g., biopolymers), such as hydrogels, agarose, gelatin, dextran, chitosan, silica gel, etc. In embodiments, the extracellular matrix may include basement membrane extracts, extracellular matrix derived from human or animal tissue or cell cultures, extracellular matrix derived from animal tissue, synthetic extracellular matrix, hydrogels, collagen, soft agar, egg white, or combinations thereof.

[0068] The extracellular matrix may also contain growth and / or differentiation substrates such as collagen, collagen I, collagen IV, fibronectin, laminin, vitronectin, D-lysine, entactin, heparan sulfate proteoglycans, or combinations thereof. In another embodiment, the matrix may contain laminin, collagen IV, entactin, and heparan sulfate proteoglycans. In some such embodiments, the matrix also contains growth factors, matrix metalloproteinases (collagenases), other proteinases (plasminogen activators), or combinations thereof. In another embodiment, the extracellular matrix may contain basement membrane extracts, extracellular matrix components, collagen, collagen I, collagen IV, fibronectin, laminin, vitronectin, D-lysine, entactin, heparan sulfide proteoglycans, or combinations thereof.

[0069] In embodiments, the extracellular matrix comprises a hydrogel. As used herein, “hydrogel” is a three-dimensional network of cross-linked hydrophilic polymer chains. Hydrogels used in cell culture may include natural and / or synthetic materials. Natural hydrogels suitable for cell culture may include protein and extracellular matrix components such as collagen, fibrin, hyaluronic acid, or Matrigel, as well as materials derived from other biosources such as chitosan, alginate, or silk fibril. Suitable synthetic hydrogels may be formed from non-natural molecules such as poly(ethylene glycol) (PEG), poly(vinyl alcohol), and poly(2-hydroxyethyl methacrylate).

[0070] In some embodiments, the extracellular matrix includes a basement membrane extract. The basement membrane is a thin extracellular matrix containing proteins and proteoglycans that lies beneath epithelial cells in vivo. Epithelial cells work with the basement membrane to form a solid barrier that protects internal life activities.

[0071] The extracellular matrix is ​​delivered to the microfluidic chip in the form of an extracellular matrix precursor. As used herein, “extracellular matrix precursor” refers to a liquid polymer or prepolymer that, upon curing, yields the extracellular matrix described herein.

[0072] After the extracellular matrix precursor is delivered, it is at least partially cured (i.e., gelled) before further liquid is introduced into the adjacent channel. In some embodiments, the extracellular matrix is ​​fully cured before liquid is introduced into the adjacent channel. Thus, a method for preparing the MPS of this disclosure comprises depositing the extracellular matrix precursor into a first channel of a microfluidic chip; and curing the extracellular matrix precursor to deliver the extracellular matrix into the first channel. The conditions used to cure the extracellular matrix precursor may vary depending on the extracellular matrix used. Any curing method suitable for a particular extracellular matrix precursor can be used. In some embodiments, curing the extracellular matrix precursor comprises solidifying the extracellular matrix precursor. In certain embodiments, curing the extracellular matrix precursor comprises solidifying the extracellular matrix precursor while simultaneously incubating the extracellular matrix precursor. The extracellular matrix precursor may be incubated, for example, at about 37°C. In other embodiments, curing the extracellular matrix precursor comprises incubating the extracellular matrix precursor with a crosslinking agent. In further embodiments, curing the extracellular matrix precursor includes ionic polymerization, thermal polymerization, or photopolymerization.

[0073] In embodiments, the extracellular matrix also includes cells (e.g., from a biological tissue sample). For example, the biological tissue may include organoids, tissue biopsies, tumor tissue, excised tissue material, or embryos. The biological tissue sample may include cells obtained from, derived from, or exhibiting a phenotype associated with a particular tissue or organ, such as nerve cells, cardiac cells, hepatic cells, renal cells, skeletal muscle cells, osteocytes, skin cells, esophageal cells, intestinal cells, gastric cells, colon cells, lung cells, or pancreatic cells. In certain embodiments, the cells are nerve cells. In some such embodiments, the nerve cells include neural progenitor cells, astrocytes, microglia, or combinations thereof derived from human induced pluripotent stem cells. The biological tissue sample may be derived from healthy or diseased tissue. Other suitable cells include human iPSC-derived cells, embryonic stem cells, and other pluripotent cells, progenitor cells, differentiateable cells, etc. For example, neurons, endothelial cells, epithelial cells, astrocytes, pericytes, cardiomyocytes, skeletal muscle cells, hepatocytes, fibroblasts, and osteocytes.

[0074] In some embodiments, cells are deposited on an extracellular matrix precursor and then cured. In some such embodiments, cells are present in the extracellular matrix precursor before being deposited in a first channel. In other embodiments, cells are introduced into or on the extracellular matrix.

[0075] As described above, the extracellular matrix precursor is cured before further liquid is introduced into the adjacent channel. Thus, in certain embodiments, a method for preparing the MPS of the present disclosure comprises curing the extracellular matrix precursor, wherein the medium is deposited in a second channel adjacent to the extracellular matrix of a first channel. Thus, in certain embodiments, a method for preparing the MPS of the present disclosure comprises curing the extracellular matrix precursor by incubating the extracellular matrix precursor, wherein the medium is deposited in a second channel adjacent to the extracellular matrix of a first channel.

[0076] In some such embodiments, the medium is confined within the volume of a channel (e.g., a second channel) adjacent to a channel (e.g., a first channel) containing the extracellular matrix. For example, in some embodiments, the medium is confined within a second volume of the second channel. In embodiments, the microfluidic chip comprises a third channel adjacent to the first channel, the third height being greater than the first height. In some such embodiments, the MPS further includes a second medium confined within a third volume of the third channel.

[0077] In other embodiments, the microfluidic chip comprises a third channel adjacent to the second channel, the third height being lower than the second height. In some such embodiments, the MPS further includes a second extracellular matrix confined within the third volume of the third channel.

[0078] In the MPS of this disclosure, an epithelial layer, also called an epithelial barrier, is positioned at the opening between adjacent channels. For example, in some embodiments, the MPS comprises an epithelial barrier positioned at a first opening between a first channel and a second channel.

[0079] In some embodiments, the epithelial barrier (e.g., the endothelial barrier) is continuous. In some embodiments, the epithelial barrier is continuous if it has 75% confluence. As used herein, “confluence” is used as an estimate of the percentage of the sidewalls of the extracellular matrix at the openings between adjacent channels that are covered by adherent epithelial cells. In some embodiments, the epithelial barrier is continuous if it has 85% confluence. In some embodiments, the epithelial barrier is continuous if it has 90% confluence. In some embodiments, the epithelial barrier is continuous if it has 95% confluence. In certain embodiments, the epithelial barrier is considered continuous if it is confluent (i.e., about 100% of the sidewalls of the extracellular matrix are covered by adherent cells).

[0080] Any suitable epithelial cells may be used. As used herein, “epithelial cells” refers to cells of epithelial origin, or cells that have been differentiated to express markers that identify cells as epithelial cells.

[0081] The cells may be animal cells (e.g., epithelial cells, cells derived from epithelial cell lines, primary epithelial cells). For example, the cells may be derived from mammals (e.g., mice, rats, dogs, cats, cattle, horses, pigs, non-human primates, and humans). In certain embodiments, the cells are human cells (e.g., epithelial cells, cells derived from epithelial cell lines, primary epithelial cells). Examples of epithelial cells that may be used include prostate cells, mammary gland cells, hepatocytes, islet cells (e.g., beta cells), lung epithelial cells, kidney cells, bladder cells, gastric epithelial cells, colonic and small intestinal epithelial cells, urethral epithelial cells, testicular epithelial cells, ovarian epithelial cells, cervical epithelial cells, thyroid cells, parathyroid cells, adrenal cells, thymocytes, gallbladder cells, and pituitary cells. Furthermore, transformed cells or established cell lines may also be used. As used herein, the term “cell line” refers to a continuously growing or immortalized cell line. In other words, a cell line is a population of cells derived from a multicellular organism that does not normally proliferate indefinitely, but which, due to mutations, can avoid normal cellular senescence and instead continue to divide.

[0082] In some embodiments, the epithelial barrier includes endothelial cells. “Endothelial cells” are cells of endothelial origin, or cells differentiated to express markers that identify cells as endothelial cells. In some embodiments, the epithelial barrier is an endothelial barrier. In some embodiments, the endothelial cells are derived from stem cells. In certain embodiments, the endothelial cells are derived from induced pluripotent stem cells. In some embodiments, the endothelial cells include hematopoietic endothelial cells. The epithelial cells forming the epithelial barrier (e.g., endothelial cells) may be obtained from or derived from the same subject from which the biological tissue sample was obtained. In certain embodiments, the endothelial barrier includes brain microvascular endothelial cells (BMECs). In certain embodiments, the endothelial barrier includes human BMECs.

[0083] Embodiments of the method for preparing the MPS of the present disclosure include culturing a continuous endothelial barrier at a first opening between a first channel and a second channel. As described above, such an epithelial barrier (e.g., an endothelial barrier) is cultured on the sidewall of the extracellular matrix after the extracellular matrix precursor has hardened and the medium has been deposited in the adjacent channel. In some embodiments, epithelial cells are deposited in the medium of the channel adjacent to the epithelial barrier (e.g., a second channel), and the microfluidic chip is tilted so that the epithelial cells settle on the sidewall of the extracellular matrix.

[0084] The medium contained in channels adjacent to channels containing the extracellular matrix is ​​selected according to the function of the channel. For example, the medium in a channel adjacent to the epithelial barrier (e.g., a second channel) is typically a growth medium that provides nutrients and oxygen after epithelial cells have been injected. Furthermore, the medium contained in such channels may be changed or replaced at various points in time.

[0085] In some embodiments, the medium of a channel adjacent to the epithelial barrier (e.g., a second channel) further comprises pericytes. In other words, in some embodiments, pericytes are included in the second channel. In such embodiments, the medium of the second channel may include a combination of endothelial cell culture medium and pericyte cell culture medium.

[0086] In some embodiments, flow is simulated through at least one channel. In various embodiments, flow is simulated by oscillating the microfluidic chip in a first direction. In such embodiments, flow is not simulated in the microfluidic chip until an epithelial barrier is established (for example, the epithelial barrier has a confluence of 75%, 85%, 90%, 95%, or 100%).

[0087] As can be understood, the MPS of this disclosure is well suited for use as an vascularized tissue model in which a medium-containing channel (e.g., a second channel) acts as a “blood” channel and an extracellular matrix-containing channel (e.g., a first channel) acts as a tissue (e.g., brain, heart, liver, kidney, skeletal muscle, bone, skin, esophagus, intestine, stomach, colon, lung, pancreas, etc.) channel. Thus, in embodiments, the MPS of this disclosure is an vascularized tissue model. In some embodiments, the vascularized tissue model is a blood-brain barrier model or a stroke model. In further embodiments, the vascularized tissue model is a heart model, skeletal muscle model, liver model, kidney model, bone model, skin model, esophageal model, stomach model, colon model, intestinal model, lung model, or pancreatic model.

[0088] In certain embodiments, the microfluidic chip of the present disclosure, when applied to the human blood-brain barrier (BBB), comprises three channels: a “blood-side” channel (i.e., the second channel, as discussed above) for simulating blood flow; a central “brain” channel (i.e., the first channel, as discussed above) for nerve cells to form a natural 3D structure within the hydrogel matrix; and a “cerebrospinal fluid (CSF)-side” channel (i.e., the third channel, as discussed above) for providing additional fluid access to the nerve cells in the “brain” channel. In preferred embodiments, the central “brain” channel is configured to be lower in height to create surface tension between the upper and lower surfaces of the channel, which works to stably contain a liquid hydrogel precursor within the “brain” channel. This embodiment of the BBB model can produce a continuous, physically intact endothelial barrier, resulting in a clearly defined boundary between the “blood-side” channel and the “brain” channel.

[0089] How to use This disclosure also provides methods for using the MPS described herein. In particular, the microfluidic devices of this disclosure and the MPS fabricated thereon may be used in methods for assaying or testing various human biological systems using these chips and models, such as BBB, NVU, CNS, blood vessels, liver, kidney, intestine, or cancerous tumors. Furthermore, the MPS may be used in 3D cell culture, co-culture, migration studies, cytotoxicity studies, and various other cell assays. In certain embodiments, the MPS of this disclosure is a vascularized tissue model. In various embodiments, the vascularized tissue model may be a heart model, skeletal muscle model, liver model, kidney model, bone model, skin model, esophageal model, stomach model, colon model, intestine model, lung model, or pancreatic model.

[0090] For example, the MPS of the present disclosure may be used in a method for screening therapeutic agents(s). In such a method, the MPS of the present disclosure is prepared as described above. After the epithelial barrier is established, the therapeutic agent is deposited into a medium in a channel adjacent to the epithelial barrier (e.g., a second channel). After a suitable period of time, the microfluidic chip is analyzed. In some embodiments, the analysis includes imaging studies. In some embodiments, cells or tissues in the extracellular matrix may be lysed and gene expression may be analyzed. Such a screening method may be used for any suitable therapeutic agent(s). For example, the therapeutic agent may be a stem cell, a small molecule, or a peptide.

[0091] For example, the MPS of this disclosure may be prepared as described above (e.g., by (1) depositing an extracellular matrix precursor containing a biological tissue sample into a first channel, (2) curing the extracellular matrix precursor, (3) depositing a medium containing endothelial cells and pericytes into a second channel adjacent to the first channel, and (4) culturing a continuous endothelial barrier on the sidewall of the extracellular matrix at the opening between the first and second channels). After the MPS is prepared and the endothelial barrier is established, the therapeutic agent is deposited into the second channel. After an appropriate period, the MPS is analyzed. For example, a microfluidic chip may be stained and imaged. Alternatively, or in addition, the extracellular matrix and embedded cells may be lysed and gene expression analyzed. Furthermore, the medium may be removed from the third channel and analyzed for concentrations of, for example, the therapeutic agent, metabolites, etc. Such a method may be repeated sequentially or in parallel with multiple therapeutic agents. In some embodiments, several iterations are performed for each therapeutic agent.

[0092] In some embodiments, the screening method is specific to a particular patient. In such embodiments, the screening results can be used to determine treatment. In certain embodiments, the cells used in the MPS of this disclosure are patient-derived cells. In some such embodiments, the pathophysiological conditions specific to individual patients are also simulated in the MPS.

[0093] In various embodiments, the MPS portion is stained before imaging. In some embodiments, a probe is introduced into the MPS before imaging. Any suitable stain (e.g., CD31, von Willebrand factor, etc.) or probe (e.g., a luminescent probe) can be used according to known protocols. Imaging studies may be used, for example, to assess neurodegeneration, to track stem cell infiltration, to record cytoplasmic calcium oscillations of neurons in ischemic stroke models, to assess the state of the endothelial barrier, etc.

[0094] kit This disclosure further provides kits for use in the preparation or use of MPS described herein (for example, in a method for screening therapeutic agents). The kits of this disclosure include a microfluidic chip, as discussed above. In some embodiments, the kits of this disclosure also include an extracellular matrix precursor or a component thereof. In certain embodiments, the kits of this disclosure include a microfluidic chip and an angiogenic compound.

[0095] Such kits may vary in the contents of any of the kits described herein, which may further include one or more reagents, assay controls, or other supplies necessary for evaluating the therapeutic agent(s), such as syringes, ampoules, vials, tubes, tubing, face masks, needleless fluid transfer devices, injection caps, sponges, sterile adhesive strips, Chloraprep, gloves, etc.

[0096] The kit may further include written instructions for using the kit in the manner disclosed herein. In various embodiments, the written instructions may include instructions for the preparation of reagents; appropriate reference levels for interpreting results related to the use of the kit; and appropriate disposal of related waste. The written instructions may be in the form of printed instructions provided within the kit, or they may be printed on a portion of the container housing the kit. The written instructions may be in the form of sheets, pamphlets, brochures, CD-ROMs, or computer-readable devices, or they may provide instructions for finding the instructions remotely, such as on a website. The written instructions may be in English and / or in a national or regional language.

[0097] Example 1 A biomimetic stroke model for systematic evaluation of the nerve repair capacity of stem cell therapy. Stem cell therapy is emerging as a promising treatment option for restoring neuronal function after ischemic stroke. Despite an increasing number of candidate stem cell types, each with unique characteristics, there is a lack of experimental platforms to systematically evaluate their neurorepair capabilities. When stem cells are transplanted into the ischemic brain, therapeutic efficacy depends primarily on the response of neurovascular units (NVUs) to these exogenous cells. We developed a biomimetic system (MPS) of ischemic stroke with functional NVUs on a microfluidic chip. The novel chip design facilitated the formation of a functional blood-brain barrier (BBB) ​​by the incorporated cells, restoring in vivo-like behavior in both healthy and ischemic conditions. Using the MPS, we tracked transplanted stem cells and characterized their neurorepair behavior, reflected in gene expression levels. Each stem cell type exhibited unique neurorepair effects, primarily by assisting endogenous restoration rather than direct cell replacement. Restoration of synaptic activity, crucial for neuronal function, correlated more closely with the restoration of structural and functional integrity in the NVU than with the regeneration of the neurons themselves.

[0098] Chip design for functional BBB reconstruction A key advantage of the in vitro model is its ability to monitor cell behavior in real time. The microfluidic chip has three channels: a “blood side” channel to simulate blood flow, a “brain” channel where nerve cells form a natural 3D structure within the hydrogel matrix, and a “cerebrospinal fluid (CSF) side” channel providing additional access to the nerve cells in the “brain” channel. On day 0, the sample was prepared by unfolding nerve tissue and injecting nerve cells into the hydrogel of the brain channel. A mixed medium of nerve growth medium and astrocyte medium was also added to the blood side and CSF side channels on day 0. On day 3, a mixed medium of nerve differentiation medium and astrocyte medium was added to the blood side and CSF side channels. On day 5, vascular development began. Vascular cells in a mixed medium of endothelial cell medium and pericyte cell medium were injected into the blood side channel, and a mixed medium of nerve differentiation medium and astrocyte medium was added by flowing into the CSF side channel. On day 10, ischemia was induced by adding glucose-free media to the blood-side channel and the CSF-side channel, and incubating the microfluidic chip with 2% oxygen. On day 11, stem cell transplantation was performed by injecting stem cells into a mixed medium of endothelial cell medium and pericyte cell medium in the blood-side channel, and adding a mixed medium of neural differentiation medium and astrocyte medium to the CSF-side channel. On day 18, the results were analyzed. This process is described in detail in Table 1.

[0099] [Table 1]

[0100] NPC: Neural progenitor cell NEM: Neuronal Growth Medium AM: Serum-free astrocyte medium ACM: Astrocyte conditioned medium NDM: Neuronal Differentiation Medium ECM: Endothelial cell medium PM: Pericyte medium For the hydrogel matrix, we used a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumors (Cultrex®, Trevigen), containing laminin, collagen IV, entactin, fibronectin, and heparan sulfate proteoglycan as its main components. The EHS tumor-derived basement membrane has an elastic modulus of approximately 0.5 kPa, within the range of physiological stiffness of brain tissue, and supports the neural differentiation of NPCs, as well as the survival and function of neurons. In previous chip designs, each microfluidic channel was separated by micropoles to generate the surface tension necessary to confine the liquid hydrogel prepolymer within the designated channels. The endothelium was supposed to form on the sidewalls of the hydrogel of the "brain" channels perpendicular to the xy plane of the entire structure. However, it was found that these micropoles interfered with the endothelial cells, preventing them from forming continuous and intact endothelium and resulting in physical defects. These deficiencies could potentially hinder the evaluation of the actual effectiveness of stem cell therapy, as they could act as artificially easy shortcuts for stem cells or bioactive substances in the bloodstream to reach ischemically damaged brain tissue.

[0101] Therefore, a new chip design was developed that, without micropoles, still possesses real-time monitoring capabilities at the same microscopic focal plane. In the new design, the height of the central "brain" channel was reduced to create surface tension between the top and bottom surfaces, allowing the liquid hydrogel prepolymer to be stably retained in the "brain" channel. A clearly defined boundary was formed between the "blood side" channel and the "brain" channel of the new chip. The reconstructed endothelium successfully prevented the free diffusion of a fluorescent probe (FITC-dextran, 4kDa) across itself. Since many pathogens, such as viruses and bacteria, are larger than 4kDa, and natural BBBs prevent these pathogens from entering the brain, a 4kDa probe size is useful for evaluating the functionality of the BBB.

[0102] The confocal microscopy image of the monolayer showed a continuous, physically intact endothelial barrier, in contrast to the barrier generated by previous chip designs with micropoles. Furthermore, the new chip design lacks micrometer-scale features, eliminating the need for soft lithography processes in chip manufacturing and enabling 3D printing.

[0103] Prior to endothelial formation, a small population of astrocytes was observed in the "blood side" channel after migration from the "brain" channel (0.9 ± 0.3 (sd)% of the total incorporated astrocytes, n=3). These migrating astrocytes, together with pericytes, assisted the BMEC, maintaining a normal, smooth, rounded morphology throughout the "blood side" channel, similar to the morphology of the BMEC when co-cultured with both astrocytes and pericytes in 2D culture. The astrocytes and pericytes in the "blood side" channel settled beneath the endothelial cell layer at the bottom as interconnected BMECs, maturing and forming endothelium. This may be due to angiogenic processes that strengthen the connections between adjacent endothelial cells through endothelial cell-intercellular junctions.

[0104] Endothelial airtightness under different conditions depending on cell composition and the presence of flow was investigated by calculating the apparent permeability coefficient. In the presence of astrocytes and pericytes, the endothelium became significantly tighter to hinder the diffusion of the probe, FITC-dextran. A further significant decrease in permeability was observed after introducing culture medium flow. This tightening of the endothelium in the presence of flow is consistent with reports of enhanced paracellular connectivity in BBBs by appropriate mechanical stimulation. The permeability coefficients of the obtained BBB models were approximately 6 × 10⁻⁶ for 4 kDa and 70 kDa FITC-dextran, respectively. -7 cm / s and approximately 8 × 10 -8 The response was cm / s, comparable to that of other previously reported in vitro and in vivo BBB models. The reconstructed BBB also exhibited the expected size-selective permeability, as in the case of a functional BBB; smaller probe sizes resulted in better diffusion across the BBB.

[0105] Another standard measure for assessing the BBB's tightness is trans - endothelial electrical resistance (TEER). TEER measurement is a simple, label - free, and non - invasive method for quantifying barrier integrity. The reported TEER values for microfluidic BBB models range from several hundred to several thousand Ωcm 2 but the permeability coefficients are within a relatively narrow range of around 1×10 -6 cm / s for 4 kDa FITC - dextran. This may be because the TEER values depend greatly on the measurement method and experimental procedures. Since direct current (DC) can damage cells, alternating current (AC) is widely used for TEER measurement. Also, four - electrode AC TEER measurement, which uses four electrodes, is more accurate than two - electrode AC measurement because it is less affected by polarization impedance at the electrode - electrolyte interface. However, due to the small surface area of the BBB on the chip, the resistance across the BBB was expected to reach several mega - ohms, exceeding the measurable range of commercially available four - electrode AC TEER meters. Therefore, two - electrode DC measurement was used, and the TEER value of the BBB on the chip was 370±20 (s.d.) Ω·cm 2 under flow. The measured TEER values were lower than those reported in some microfluidic BBB models, but showed a significant difference between conditions.

[0106] Once the physical integrity of the endothelium was confirmed, the functional properties of the reconstructed endothelium as a biochemically intact barrier were investigated. One of the key functions of the brain endothelium in vivo is to isolate neurons in the brain parenchyma from any pro-inflammatory substances in the bloodstream. To maintain the original phenotype of the cells in each channel of the chip, two different types of media were deployed: serum-containing endothelial media for the "blood-side" channel and serum-free glial cell media for the "CSF-side" channel. The reason for this setup is that endothelial cells require serum to maintain their original phenotype in vitro, while glial cells exhibit pro-inflammatory behavior in serum-containing culture media. Serum extracted from whole blood is an undefined mixture of proteins, hormones, minerals, growth factors, and lipids. Therefore, the reconstructed BBB needs to prevent the entry of any pro-inflammatory substances from serum in the "blood-side" channel. In samples without the BBB, microglia, a type of commensal immune cell in the brain, exhibited pro-inflammatory behavior as expected due to direct exposure to serum. In contrast, in samples with reconstructed blood-brain barriers (BBBs), microglia did not exhibit such pro-inflammatory behavior, and the chip's BBBs were confirmed to be biochemically intact barriers, similar to natural BBBs.

[0107] To be a clinically important platform for stem cell therapy, the BBB of the chip should also exhibit different responses based on the phenotype of the invading cells. While the neurorepair effect of each stem cell type may depend on their ability to invade across a narrow BBB and reach the lesion site, little is known about the innate BBB response to candidate stem cell types in therapy. Therefore, as a valid measure to demonstrate the cell-selective responsiveness of the BBB, we used the well-established metastatic behavior of two human breast cancer cell lines, MB-231 and its brain metastatic derivative population, MB-231Br. MB-231Br specifically invades across the BBB and shows a much stronger tendency to metastasize than MB-231 in animal models. The reconstructed BBB exhibited the expected cell-specific response to these two types of invasive cancer cells, confirming its in vivo-like functionality and validating its sensitivity to the phenotype of the invading cells.

[0108] Establishment of ischemia After confirming the formation of a functional blood-brain barrier (BBB) ​​in the chip, ischemic conditions were established. Ischemic injury has two main zones: the core infarct zone and the ischemic penumbra (also called the periinfarct rim). The core infarct zone is characterized by a lack of blood supply and severe necrosis of nerve cells, and is considered to be irreversibly damaged. In contrast, the ischemic penumbra, the rim surrounding the irreversibly damaged core, has a blood supply sufficient for cells to survive but not enough to properly communicate and function. This periinfarct rim is considered a therapeutic target for recovery after stroke. Therefore, the goal was to recreate this periinfarct zone, sufficiently damage cells, and establish ischemic conditions that minimize cell death.

[0109] The optimized ischemic condition was 2% O2 with serum and glucose depleted for 24 hours in the absence of flow. This ischemic condition induced detectable cytotoxicity, measured by the amount of extracellular lactate dehydrogenase (LDH) released through the damaged cell membrane, while preserving cell viability. We also observed that hypoxia-inducible factor-1α (HIF-1α), normally found in the cytoplasm of cells under normal oxygen conditions, translocated to the nucleus, as observed in ischemic brains in vivo. Based on the pattern of gene expression changes, ischemic injury upregulated genes in both apoptosis and anti-apoptotic signaling cascades, as has just been reported in animal ischemic stroke models. Redox reactions were also upregulated, meaning that cells protected themselves against the increased intracellular levels of reactive oxygen species in ischemia. Upregulation of neurotrophic and angiogenic factors suggests that ischemic-injured cells attempt to repair and remodel themselves. The cells also exhibited a typical neuroinflammatory response to ischemic stroke, as indicated by the upregulation of gene expression for pro-inflammatory cytokines and integrins. Downregulated expression of extracellular matrix proteins, along with enhanced matrix metalloproteinase activity and reduced interaction between cells and the ECM, suggests that ischemic injury led to impaired tissue integrity and subsequent tissue remodeling processes. Collectively, these gene expression patterns indicate that the ischemic condition successfully induced inflammation and decreased tissue integrity as expected, accompanied by endogenous neuroprotection and tissue remodeling, as reported in many other in vivo stroke models.

[0110] Verification of NVU behavior To validate the functionality of reconstructed NVUs, we investigated the behavior of individual cells at various levels under both healthy and ischemic conditions. At the genetic level, we measured changes in gene expression associated with a range of post-stroke pathological states and classified them based on their functional characteristics. Since most genes are not cell-specific and are involved in multiple cellular processes, this grouping is solely for the purpose of outlining the overall pattern of responses across cell populations in the experiment.

[0111] Neurons are the primary components of the central nervous system and play a crucial role in neuronal function. Considering the short lifespan and limited proliferative capacity of primary human neurons in vitro, we used neural progenitor cells (NPCs) derived from human iPSCs (induced pluripotent stem cells) in a stroke model, optimizing the culture conditions of the chips for their neuronal differentiation. Differentiated NPCs exhibited neuronal morphology in cell body and axonal branching and dendrites, and expressed mature neuronal markers such as microtubule-associated protein 2 (MAP-2), a family of proteins that regulate neurotransmitter release at synapses, as well as synapsin I and II (SYN). They also maintained close resemblance to astrocytes. Under ischemic conditions, they showed beading or fragmentation of degenerated dendrites, a typical morphology of degenerated neurons, compared to the smooth, clear dendritic morphology observed under normal oxygen conditions. They were also stained with the neurodegenerative marker Fluoro-Jade, consistent with reports from in vivo ischemic stroke models.

[0112] Ischemic changes in gene expression indicate that endogenous repair resulted in upregulation of genes involved in neurite formation and synapse formation, but accompanied by downregulation of genes related to synaptic plasticity. Simultaneously with hyperstimulation of the excitatory neurotransmitter glutamate, decreased activity of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) (ABAT and GABRB1 are encoded by an enzyme and a GABA receptor, respectively, for GABA catabolism) was also observed. These expression patterns suggest a disrupted balance between neuronal excitation and inhibition under ischemic conditions, potentially leading to the excitotoxicity typically observed in ischemic stroke.

[0113] Cytoplasmic calcium (Ca) in differentiated NPCs 2+ We also investigated how ischemic conditions are reflected in the oscillation patterns. 2+Imaging provides an indirect but accurate measure of action potential generation in individual neurons, representing a range of neuronal functions from synaptic activity to intercellular communication, adhesion, neurodegeneration, and apoptosis. 2+ The image shows differentiated NPCs with cytoplasmic Ca 2+ Four typical signal patterns: Oscillatory (free Ca 2+ Repeated short-term increases, transient (Ca through membrane calcium channels) 2+ (Short-term increase due to inflow), persistence (Ca due to both external and internal storage) 2+ This indicates a sustained increase in Ca levels, or a less pronounced signal. Ischemic injury may indicate a less pronounced Ca level. 2+ The proportion of cells showing signaling was decreased, while the proportion of cells showing both transient and sustained signaling was increased. Ca accumulated in the cytoplasm. 2+ The level is thought to lead to neuronal death in animal stroke models. Analysis of oscillatory signal changes due to ischemia revealed a slight change in amplitude, but a significant increase in oscillatory frequency, and Ca to cells. 2+ This indicates an increase in inflow. 2+ Excessive influx of this substance, along with a disrupted balance between neuronal excitation and inhibition, indicates excitotoxic neurodegeneration in ischemic samples.

[0114] Brain microvascular endothelial cells (BMECs) are the major cellular components of the cerebral vascular system BBB. Human primary BMECs were used throughout the study. Compared to endothelial cells found in other tissues, BMECs have high mitochondrial density, lack of fenestration, low pino cell activity, and high density adhesion and tight junctions. Tight junctions determine the paracellular airtightness of endothelial cells and their permeability across the BBB. Closure zone-1 (ZO-1) is a major junction adapter protein that regulates other junctional elements, intercellular tension, angiogenesis, and BBB formation. Flow through the "blood side" channel increased ZO-1 expression and elongated the cell body shape along the simulated direction of blood flow. Upregulated ZO-1 expression in samples with flow resulted in upregulation of the expression of other junction proteins, VE-cadherin and claudin-5. ZO-1 expression in ischemic samples was significantly reduced compared to normoxic samples with flow, but was statistically comparable to normoxic samples without flow. Importantly, ZO-1 expression, which is primarily localized to the cell membrane under normal oxygen conditions, spreads throughout the cell body under ischemic conditions. This dispersed spatial distribution of ZO-1 in ischemic samples results in increased permeability of a fluorescent probe (4kDa FITC-dextran), representing decreased paracellular airtightness under ischemia. These results suggest that paracellular airtightness between endothelial cells is more significantly influenced by the degree of tight junction localization of the cell membrane than by the overall level of their expression. BMEC in ischemic samples significantly increased the expression of vascular endothelial growth factor (VEGF), one of the angiogenic factors, suggesting that post-stroke vascular reorganization occurred, as observed in animal stroke models. At the genetic level, ischemic injury reduced paracellular connectivity but upregulated genes involved in vasoconstriction and adhesion molecules for mobilizing immune cells, as observed in animal stroke models.

[0115] BMEC behavior has been well documented under various experimental conditions. In monocultures of human BMECs, flow-induced shear stress did not significantly affect the expression or morphology of tight junction proteins. On the other hand, flow conditions in monocultures of bovine BMECs resulted in upregulation of tight junction proteins and morphological alignment along the flow direction. In another in vitro study, rat BMECs required appropriate interactions with both astrocytes and pericytes, exhibiting tight junction localization of their original patterns around the cell membrane, as observed in this model. Taken together, these results suggest that for human BMECs to exhibit in vivo-like behavior, several key components of the original BBB microenvironment—mechanical stimulation by blood flow and the heterocellular network of NVUs—are necessary. This stroke model provides both of these microenvironmental features, enabling in vivo-like behavior of human BMECs.

[0116] Pericytes are parietal cells of the microvascular system that regulate BBB permeability, angiogenesis, clearance, cerebral blood flow, neuroinflammation, and stem cell activity. Human primary cerebral vascular pericytes were used. Pericytes in the chip expressed platelet-derived growth factor receptor beta (PDGFRβ), one of the pericyte-specific markers, and were located between the mature endothelium and the lateral wall of the "brain" channel. Pericytes were activated in response to ischemic injury and contributed to vascular inflammation. The interaction between pericytes and endothelial cells was critical to vascular stability under normal conditions and was downregulated.

[0117] Astrocytes are the primary glial cell type in the brain and play many mediating roles in heterocellular interactions in the NVU. Human primary astrocytes were used throughout this study. One of their roles is to sense neuronal metabolic activity and regulate vasodilation and vasoconstriction to match blood flow accordingly. Astrocytes play these intermediate roles through interactions based on direct contact with endothelial cells. Oxygen and nutrients are supplied only through “blood side” and “CSF side” channels, and as a result, astrocytes of the “brain” channel must migrate toward the endothelial layer formed at the boundary to access nutrients, extending their terminal feet and thus forming physical contact with it. This physical contact was indirectly confirmed by immunofluorescence staining of water channel proteins encoded by aquaporin-4 (AQP4), the most abundant water channel in the brain. Water channels in astrocytes are localized around the terminal feet of astrocytes that are in direct contact with blood vessels under normal conditions. This polarized location reflects their mediating role in gas exchange, including O2, CO2, and NO. In inflammatory conditions such as ischemia, the immunoreactivity of AQP4 in astrocytes is impaired from the outset, signifying a disruption of the mediating role of AQP4. Furthermore, astrocytes from ischemic samples exhibited reactive astrogliosis characterized by abnormal hypertrophy, massive proliferation, and upregulated glial fibrillary acidic protein (GFAP) expression levels. Astrocytes were unable to exhibit these behaviors under conventional 2D culture conditions. Gene expression patterns reveal a heterogeneous population of astrocytes mixed with both A1 (pro-inflammatory) and A2 (ischemic) phenotypes, as reported in in vivo stroke models. Activated astrocytes then reduced their nutritional support to neurons in ischemic stroke models, consistent with reports from other stroke models.

[0118] Microglia are commensal macrophages and the only immune cell type in the brain. Due to issues with reliable batch-to-batch reproducibility in primary human microglia, transformed human microglia cell lines (HMC3, ATCC) were used. Brain microglia exhibit an immediate pro-inflammatory response to any injury or infection. When activated, their pro-inflammatory morphological changes manifest as contraction and thickening of the process, as well as hypertrophy of the cell body, which was successfully reproduced in ischemic samples. They also rapidly secrete interleukin-1β (IL-1β), one of the pro-inflammatory (M1) phenotypic markers, within hours of inflammation onset. However, this upregulation of IL-1β is transient and not sustained. On the other hand, the expression of differentiation cluster 68 (CD68) and ionized calcium-binding adapter molecule 1 (IBA-1) is upregulated during the M1 phase and persists throughout the subsequent anti-inflammatory (M2) phase. These in vivo-like temporal patterns of IL-1β and CD68 immunoreactivity were observed in a stroke model. In contrast, conventional 2D culture conditions failed to induce these behavioral changes in microglia. Gene expression patterns indicated that ischemic onset resulted in upregulation of both pro-inflammatory (M1 phenotype) and anti-inflammatory (M2a and M2b phenotypes) microglial markers, as observed in in vivo studies. Both M2a and M2b are involved in phagocytosis and produce anti-inflammatory cytokines, but their activation signaling pathways differ from each other. In contrast, the M2c phenotype, usually considered a marker of the inactivation stage, was barely present in the 24-hour timeframe after ischemic onset in the stroke model, consistent with reports that M2c macrophages appeared only after downregulation of inflammation. Many other immune receptors and chemoattractants also showed upregulated expression levels. As previously reported, genes involved in both innate and adaptive immune responses were generally upregulated under ischemic conditions. While gene expression of purine receptors, which are involved in both immune cell regulation and neurogenesis, appeared rather inconsistent, it was also clear that the overall immune response was not adequately regulated immediately after ischemia.These changes in gene expression indicate that ischemic injury induced a wide-ranging immune response, from exacerbation of ischemic injury to aiding repair, as observed in other ischemic stroke models.

[0119] Characterization of the nerve repair capacity of stem cells While a considerable number of studies support the neurological repair capabilities of stem cells for stroke treatment, some reports contradict some of these observations. This may be partly because experiments were all conducted under different conditions and / or focused on different aspects of the complex recovery process. Being an in vitro system, this stroke model allows for identical experimental conditions across numerous samples and replicates. Therefore, it serves as a valuable platform for systematically investigating the neurological repair capabilities of clinically relevant stem cells. Stem cells examined in this stroke model include neural progenitor cells (hNPCs) derived from human induced pluripotent stem cells, neural stem cells (hNSCs) derived from human embryonic stem cells, human hematopoietic stem cells (hHSCs), mesenchymal stromal / stem cells (hBMSCs) derived from bone marrow, mesenchymal stromal / stem cells (hAMSCs) derived from adipose tissue, and endothelial cell progenitor cells (hEPCs). The effects of reperfusion treatment alone without stem cells were investigated by reintroducing oxygen and glucose after ischemic injury.

[0120] Neuronal repair after ischemic stroke involves a broad range of processes, from neuronal regeneration and immunosuppression to vascular structure repair and restoration of heterocellular interactions in NVUs. 123 relevant genes involved in each of these aspects were selected based on experimental data on ischemic responses in human neurodevelopmental PCR arrays (Qiagen) and chips. Overall gene expression in the selected set was generally upregulated only by the incorporation and reperfusion of all stem cell types. Considering genes with more than fourfold changes in expression, hNPCs and hNSCs were associated with largely strongly upregulated genes, while the opposite was true for hBMSCs. hEPCs found nearly equal numbers of strongly upregulated and downregulated genes.

[0121] Stem cell uptake and reperfusion generally had a positive effect on cell generation (neurogenesis) in the nervous system, although the degree of this effect differed depending on the stem cell type. Furthermore, all groups enhanced the expression of genes involved in neuronal migration, neuronal differentiation, neuronal fate constraint, axon formation, and glialization, while hBMSCs also showed inhibitory effects on neuronal differentiation and neuronal fate constraint. Notably, reperfusion upregulated the expression of all genes involved in neuronal migration, albeit to a weaker degree. Regarding synaptic responses, similar patterns were revealed in the regulation of synaptic organization and synaptic plasticity, with the exception of the fact that hEPCs and reperfusion showed equally strong enhancing and inhibitory effects, and all experimental groups showed a positive influence on the dominance.

[0122] In the post-stroke recovery process, suppressing inflammation initiated by ischemia is also crucial. hNPCs and hNSCs most strongly upregulated inflammation-related genes. hAMSCs also slightly upregulated, but hAMSCs, hBMSCs, hHSCs, and perfusion slightly repressed genes in the inflammatory response group. To investigate more specific inflammatory responses, we measured the expression of glial phenotype markers and examined the effect of transferred stem cells on their inflammatory behavior.

[0123] Regarding the effects on BMECs, hNPCs, hNSCs, and hEPCs upregulated the expression of tight junction protein 1 (TJP1), while the expression of claudin 5 (CLDN5) was downregulated in all groups, despite the fact that TJP1 and CLDN5 closely interact to form the blood-brain barrier (BBB). The expression of PECAM1, one of the endothelial adhesion molecules involved in the recruitment of immune cells after brain injury, was effectively downregulated in all groups. Regarding the effects on pericytes, only hAMSCs suppressed the expression of CSPG4, a reactive pericyte marker. The expression of CD248, which is involved in the role of pericytes in mediating angiogenesis, was upregulated only by hNSCs and hAMSCs and suppressed generally by the rest. Regarding the effects on microglial activity, none of the groups suppressed the expression of CD68, a microglial responsiveness marker that is normally upregulated throughout the inflammatory phase. In contrast, CD86, a pro-inflammatory M1 phenotypic marker, was successfully suppressed by all groups except hAMSCs and hHSCs. hHSC enhanced the expression of CD206, an anti-inflammatory M2a phenotypic marker, while hEPC and hBMSC promoted the expression of CD32a, an anti-inflammatory M2b phenotypic marker. hBMSC, hAMSC, and reperfusion upregulated the expression of CD163, a microglia inactivation phenotypic marker. Regarding effects on astrocytes, no group suppressed the expression of VIM, a panreactive astrocyte marker. hNPC was the only group that successfully suppressed the expression of C3, an A1 (inflammatory) reactive astrocyte marker. In all groups, the expression of CD109, an A2 (ischemic) reactive astrocyte marker, was downregulated. Taken together, the expression patterns of these astrocyte-reactive markers suggest that 7 days after ischemic injury, there was little impact from ischemia itself, and yet astrocytes still retained their inflammatory behavior. Expression of IFITM3, which is involved in neurotrophic support of astrocytes, was upregulated in all groups, while expression of FABP7, another gene with a similar function, was upregulated only by hNPC, hEPC, and hBMSC.The complexity of the overall gene expression patterns suggests that all six types of stem cells and reperfusion have their own unique pathways for suppressing ischemia-induced neuroinflammation.

[0124] When gene expression patterns were hierarchically clustered, hNPCs and hNSCs stood out as distinct groups, exhibiting more restricted fate constraints on neurons. Differential expression analysis between the two groups (those with neuronal differential volume (NDC) versus those without NDC) identified 27 genes. GO enrichment analysis of the identified genes, based on the STRING database, revealed that stem cells with NDCs are beneficial in neurogenesis and other closely related GO terms. Stem cells with NDCs also had a positive effect on regulating the signaling cascade of mitogen-activated protein kinase (MAPK), a key regulator of ischemic and hemorrhagic cerebrovascular diseases.

[0125] To better distinguish the neurorepair properties of each stem cell type, GO enrichment analysis was performed, focusing on genes with more than fourfold expression changes after stem cell uptake, to identify the primary therapeutic pathways for each stem cell type. hNPCs showed the greatest potential for neurogenesis, particularly neuronal generation, but they also had stimulating effects on other repair functions. The effects of hNSCs spread more uniformly across diverse aspects, including tissue structure formation and maturation, as well as the development of multicellular organisms. hNSCs also showed a strong ability to generate blood vessels, which are the basic environment for repairing NVU function, and to promote cell migration, a key feature for reorganizing ischemically damaged structures. Other relative advantages of hNSCs included enhanced adaptation to environmental changes and better regulation of immunity. Notably, hNSCs strongly suppressed acute inflammatory responses. hNSCs were also the least associated with pathways in cancer, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Across the entire range of GO terms, hEPCs showed a tendency toward simultaneous promotion and inhibition, while hBMSCs consistently inhibited most of them.

[0126] Compared to the top three most influential stem cell types (hNPC, hNSC, and hEPC), the remaining experimental groups (hBMSC, hAMSC, hHSC, and reperfusion therapy) induced relatively small changes in gene expression. GO enrichment analyses were also performed on these groups for genes exhibiting more than twofold changes in expression. Unlike the clear characteristics revealed in previous analyses of the three most influential stem cell types, these groups showed relatively inconsistent effects on GO terms grouped together for their relevant functions. Some consistent trends included the promotion of neurogenesis by hHSCs and the inhibition of vascular development by hAMSCs and reperfusion therapy.

[0127] Synaptic activity was further investigated as a crucial and reliable parameter for estimating the degree of recovery after stroke. Based on GO enrichment analysis of genes with at least 4-fold or 2-fold expression changes, the significance of synapse and neurotransmitter-related GO terms was compared to examine the extent to which each stem cell type promoted synaptic activity. This analysis also revealed the nature of the strong positive effects of hNSCs in many aspects of nerve repair at the synaptic level: synaptic organization and synaptic transmission, as well as the modulation of transmission pathways for relevant neurotransmitters such as glutamate, acetylcholine, and GABA. Given the molecular and functional complexity of synapses and the importance of their coordination in nerve function, hNSCs stand out as a highly promising therapeutic agent among all the stem cells evaluated.

[0128] Tracking transplanted stem cells While the precise mechanisms underlying the neurorepair effects of transplanted stem cells in stroke patients remain unclear, there is growing evidence that the therapeutic effects of stem cell therapy are mediated by indirect mechanisms such as the release of trophic factors and immunomodulatory cytokines, promotion of endogenous stem cell migration, and enhancement of endogenous neural plasticity and functional recovery. However, there are rare reports of transplanted stem cells directly replacing host cells and reconstructing damaged neural circuits. The main factors attracting stem cells to the infarcted brain parenchyma appear to be the inflammatory response of NVU, including the upregulation of cytokines, CAMs, and MMPs, which are also observed in this NVU model.

[0129] This stroke model allowed us to track transplanted stem cells and assess the extent of direct cell replacement. We examined each of the key indicators of cell replacement: the degree of attachment to the blood-brain barrier (BBB), the number of viable cells, the degree of infiltration into the "brain" channel, and differentiation into various neuronal cell types in the non-vasoconstrictive nucleus (NVU). First, we prepared GFP-expressing stem cells using lentiviral factors. The number of stem cells that initially attached to the BBB was, in most cases, less than 5% of the total number of cells in the chip. Seven days after stem cell injection, the viability of those that bound to the BBB was generally decreased (hNPCs, hNSCs, and hHSCs) or slightly increased (hBMSCs and hAMSCs). hEPCs, in contrast, proliferated vigorously and infiltrated the "brain" channel. At the same point in time, seven days after transplantation, all of these transplanted stem cells expressed little of their stem cell markers that they originally expressed in 2D cultures. This does not mean that they had successfully completed differentiation by that point, as only a very limited number of cells had matured enough to express their predicted lineage markers. Only hNPCs and hNSCs showed detectable neuronal differentiation, and even those accounted for less than 0.01% of the total number of cells in the chip. This extremely limited stem cell differentiation suggests that direct cell replacement is not the primary mechanism underlying the neurological repair effects of stem cell therapy, and this is supported by the growing evidence to that effect.

[0130] Consideration The data revealed three key aspects of the NVU microenvironment necessary for the in vivo-like behavior of constituent cells: the formation of an intact BBB, a heterocellular network, and appropriate mechanical stimulation by blood flow. The brain-like microenvironment ensures that the cells in this model retain their innate behavior and exhibit clinically important responses to ischemic injury. This model served as an efficient screening platform for investigating the neurological repair capabilities of stem cells for use in preclinical studies. We systematically analyzed how each type of stem cell affected gene activity during the complex disease progression and recovery process. We also used this stroke model to track the behavior of stem cells transplanted into ischemically damaged NVUs.

[0131] This microfluidic chip design is well-suited to the need to establish a functional blood-brain barrier (BBB) ​​and simultaneously enables real-time monitoring of transplanted stem cells migrating across the BBB. Similar chip designs have been proposed to construct a functional BBB: by positioning cells next to each other using micropoles (AIM Biotech) or flow guide structures (PhaseGuide® technology, Mimetas). Such chip designs are useful for observing the behavior of drugs or cells passing through the BBB in a 3D environment.

[0132] What distinguishes this design from prior art designs, such as those using phase guides or porous membranes, is the absence of physical structure between two adjacent channels, thereby enabling cell interactions without any potential interference caused by physical structure.

[0133] Stroke models successfully revealed the neural repair behavior of each candidate stem cell type for stroke treatment. The advantages of hNPCs and hNSCs, stem cells with the ability to differentiate into neurons, consistently stood out in many aspects related to the post-stroke recovery process. iPSC-derived hNPCs (Millipore, catalog number: SCC035) were tested by the manufacturer to ensure that over 80% of their offspring reliably differentiate into neurons. The hNSCs used in this study were initially isolated from fetal cortical brain tissue (M031 clone) at 13.5 weeks of gestation and classified as neural stem cells due to their ability to self-replicate and produce offspring cells that differentiate into neurons. Based on GO analysis, hNPCs showed the strongest ability in neuronal generation, while hNSCs showed a compelling positive effect on overall structural and functional integrity in the NVU. Notably, the recovery of NVU functionality, including glialogenesis, angiogenesis, and immune system processes, was also associated with enhanced synaptic activity mediated by hNSCs. Given the importance of synaptic activity in the rewiring of neural networks and neural function, these results suggest that restoring the functionality of the entire NVU may be more important for stroke treatment than simply replacing the neurons themselves. When interpreting these results, it is also important to consider the limitations of this approach. First, efficacy evaluation focused only on the gene level, as represented by GO functional analysis, and did not cover the full range of interactions across different levels associated with post-stroke recovery. Transcriptomics were also performed on the whole cell population, which has limitations in showing cell or tissue-specific changes. There is also always a risk of overinterpretation of GO analysis results. Second, contributions from peripheral immune cells crossing the BBB were not addressed in this model and may also play a significant role in post-ischemic inflammation. Since the incorporated neurons differentiated from neural progenitor cells rather than mature neurons, the chip may have contained a subset of neurons with heterogeneous maturity. Third, since the flow within the chip was bidirectional, induced by the oscillating shaker, endothelial cells may have activated different signaling pathways of mechanotransmission compared to unidirectional blood flow in vivo.

[0134] The results of tracking transplanted stem cells suggest that the therapeutic effect of stem cells arises not from direct cell replacement, but primarily through indirect mechanisms supporting endogenous restoration. At the time of gene expression change analysis, the number of stem cells remaining in the sample was mostly less than 1% of the total cell population. The mere presence of such a small population could not have been the primary driving force to induce the observed degree of ploidy change in gene expression across the total cell population. This means that the presence of the remaining stem cells itself had only a slight impact on the efficacy assessment. Similar observations have been reported in both animal models and clinics that transplanted stem cells hardly reached the ischemic area but still induced a significant therapeutic effect. Based on these observations and implications, preclinical evaluation of candidate stem cells for cell therapy would be more effective and important if we focus on their ability to repair damaged NVUs, both structurally and functionally, rather than tracking the fate of the transplanted stem cells themselves in vivo.

[0135] Many previous studies have presented conflicting results regarding not only the nerve repair capabilities of each stem cell type under various conditions, but also the mechanisms by which stem cells exert their therapeutic effects. Possible reasons for this controversy include the fact that efficacy evaluations have focused on only a few aspects, lacking a comprehensive analysis of the overall recovery process. Another reason may be the comorbidities often associated with stroke, such as hypertension, high cholesterol, and diabetes, which complicate disease progression and treatment. Therefore, stem cell therapy would be most effective using a personalized approach based on the comprehensive health status of each individual patient. In vitro stroke models, as described herein, would serve as an ideal platform for developing personalized stem cell therapies by utilizing patient-derived cells and simulating the unique pathophysiological state of each patient. Personalized stroke models would then serve as an efficient testing ground for screening many different candidate stem cells and identifying the optimal stem cell regimen for a given patient. Multi-omics approaches, as presented in some recent studies, can further broaden our understanding of the post-stroke nerve repair process, and in vitro stroke models are readily applicable to this purpose as well.

[0136] In summary, this approach successfully replicated the behavior of NVUs under normal and ischemic conditions in vitro, enabling efficient and systematic evaluation of stem cell therapy and overcoming the limitations of both animal models and currently available in vitro models. These findings, particularly the characterization of the nerve repair capabilities of various stem cells, can guide the direction of advanced stem cell therapy in research and clinical settings. The presented experimental platform is also immediately applicable to a wide range of other diseases related to the vascular system, opening up new possibilities in the field of precision medicine.

[0137] method cell culture NVU constituent cells Human primary astrocytes (ScienCell, catalog number: 1800) were cultured in astrocyte medium (AM) (ScienCell, catalog number: 1801) in T75 pre-coated flasks containing 2% poly-L-lysine solution (Sigma). Transformed human microglia cell lines (HMC3, ATCC, catalog number: CRL-3304) were maintained in Eagle's Minimal Essential Medium (EMEM, ATCC) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (hNPC) (Millipore, catalog number: SCC035) were maintained in NEM in T75 pre-coated flasks containing 1% Matrigel (BD Matrigel Matrix High Concentration). Human primary brain microvascular endothelial cells (BMECs) (ScienCell, catalog number: 1000) were cultured in endothelial cell medium (ECM) (ScienCell, catalog number: 1001) in T75 pre-coated flasks containing 2% collagen solution (Sigma). Human brain vascular pericytes (ScienCell, catalog number: 1200) were grown in pericyte medium (PM) (ScienCell, catalog number: 1201) in T75 pre-coated flasks containing 2% poly-L-lysine solution (Sigma). T75 flasks coated with Matrigel and those coated with poly-L-lysine solution were prepared by incubation at 37°C for 1 hour and overnight, respectively. Collagen-coated T75 flasks were prepared by incubation at 4°C overnight.

[0138] stem cells Human endothelial progenitor cells (hEPCs) were purchased from Celprogen (San Pedro, catalog number: 37089-01) and grown in complete hEPC growth medium (Celprogen, catalog number: M36053-05ES) in T75 flasks (Celprogen, catalog number: E36053-05-T75) pre-coated with extracellular matrix for hEPC growth. Human bone marrow-derived mesenchymal stem cells (hBMSC, Gibco, catalog number: A15652) and human adipose-derived mesenchymal stem cells (hAMSC, Gibco, catalog number: PCS-500-011) were maintained in mesenchymal stem cell medium (MSCM) (ScienCell, catalog number: 7501). Human neural stem cells (hNSCs, NR1) derived from the embryonic stem cell line H9, initially isolated from fetal cortical brain tissue at 13.5 weeks of gestation (M031 clone), were cultured under the same conditions as hiPSC-derived NSCs. Human hematopoietic stem cells (hHSCs) were purchased from ATCC (catalog number: PCS-800-012) and used directly in the experiment without subculturing. The culture medium was changed every 2-3 days. Cells were subculturized when confluence reached approximately 80%. Transformed microglia, hBMSCs, and hAMSCs were subculturated using 0.25% trypsin-EDTA. Astrocytes, BMECs, pericytes, hEPCs, and hNSCs were detached using 0.05% trypsin-EDTA. hNPCs were subculturated using StemPro® Accutase® cell dissociation reagent (Gibco, catalog number: A1110501).

[0139] Design and manufacturing of microfluidic chips A master mold for the microfluidic chip was fabricated using a stereolithography 3D printer. The printed mold was thoroughly cleaned with 99% isopropyl alcohol to remove unreacted monomers and curing agents, and incubated overnight on a hot plate at 50°C in a UV light chamber (wavelengths: 365nm and 405nm, power: 48W). This cleaning process was repeated for at least 3 days before being used for chip fabrication. The surface of the mold was then spray-coated with a silicone release agent (CRC, catalog number: 03300), and PDMS (Sylgard182, Dow Corning) was poured over it. After thermal curing at 65°C for approximately 5 hours, the solidified PDMS replica was peeled from the mold. A biopsy punch was used to create holes (1.5 mm in diameter) at both ends of each channel of the PDMS replica. The PDMS replica was then bonded to a pre-washed microscope glass slide (Fisher Scientific) by plasma treatment (Harrick Plasma, catalog number: PDC-32G). Before cell seeding, the microfluidic chip was UV-treated overnight for sterilization.

[0140] Rebuilding functional NVU chips Reconstruction of brain tissue To construct functional brain tissue on a microfluidic chip, human iPSC-derived NPCs, astrocytes, and microglia were embedded in a basement membrane extract (BME) hydrogel (Cultrex®, Trevigen, catalog number: 3433-001-R1, with reduced R1 type growth factor basement membrane matrix) and then injected into the "brain" channel of the chip. hNPCs were suspended in nerve growth medium (NEM, Millipore, catalog number: SCM004) supplemented with 2 mM glutamine and 0.02 μg / mL FGF-2. To obtain resting astrocytes and microglia, they were maintained for one day prior to injection in serum-free AM and astrocyte-conditioned medium (ACM, ScienCell, catalog number: 1811), respectively. The density of the suspensions for each cell type was approximately 8 × 10⁶. 6The cell-cell ratio was 4 × 10¹⁶ cells / mL. A cell mixture was prepared by mixing hNPCs, astrocytes, and microglia in an 8:4:1 (n / n / n) ratio, and then mixing with BME type R1 hydrogel prepolymer (gel:cell = 4:1 (v / v)). According to the vendor, over 80% of hiPSC-derived NPCs commit to mature neurons, and the final cell ratio for neurons, astrocytes, and microglia ranges from 5 to 6:4 to 5:1 (n:n:n), similar to a naive brain. The gel-cell mixture was injected into the "brain" channel of a chip placed on a cold pack. The total number of neurons incorporated into the "brain" channel was 4 × 10¹⁶. 4 The procedure was as follows: After injection, the chips were transferred to rectangular 4-well cell culture plates (Thermo Scientific, catalog number: 267061) and incubated in a cell culture incubator at 37°C for 30 minutes for gelation. After gelation, a serum-free mixed medium of NEM, serum-free AM, and ACM (8:4:1, v / v / v) (referred to as NEM / AM) was injected into both the "blood side" and "CSF side" channels and then replaced daily. From day 3 after injection, NEM was replaced with a neuronal differentiation medium (NEM) (Millipore, catalog number: SCM111) called (NEM / AM). The culture medium was replaced every other day for the next two days until BBB reconstruction.

[0141] BBB reconstruction BMEC and human pericytes, approximately 1 × 10⁶ 6 Cells were suspended in ECM and PM at a density of cells / mL, respectively. BMEC and pericytes were mixed in a 9:1 (n / n) ratio based on the literature, and nerve cells were co-cultured in the "brain" channel for 4 days. Then, 10 μL of the cell suspension was injected into the "blood side" channel of the tip. The total number of cells in the "blood side" channel was 1 × 10⁶. 4The procedure was as follows: The chip was tilted slightly so that BMECs and pericytes adhered to the sidewall of the hydrogel of the “brain” channel, and it was incubated for 3 hours. The old medium was then removed, and fresh mixed medium (ECM:PM = 9:1 (v / v), final serum content 4.7% (v / v) (referred to as ECM / PM)) was injected into the “blood side” channel to remove any unadhered cells and debris. The mixed medium of ECM and PM in the “blood side” channel, as well as the mixed medium of NDM, AM, and ACM in the “CSF side” channel, was changed every other day. The chip was cultured for a further 3 days for BBB formation. In experiments tracking pericytes, BMECs and pericytes were pre-stained with DiD and DiO cell labeling solutions (Vybrant®, Invitrogen), respectively, and their whole nuclei were counterstained with NucBlue® Live ReadyProbes® reagent (Themo Fisher Scientific).

[0142] Shear stress of BBB Shear stress in physiological flows such as the cerebral microvascular system (0.01~10 dyne / cm²) 2 To apply this, a pulsating bidirectional flow was generated by placing the sample on a rocking seesaw shaker (Mimetas, OrganoFlow® L). The model's design parameters were adjusted based on the following equation:

[0143]

number

[0144] Here, τ = shear stress (dyne / cm) 2 ), Q = flow rate (cc / s), μ = viscosity of culture medium, b = channel width, and h = channel height. Based on Poiseuille's principle,

[0145]

number

[0146]

number

[0147] (The hydraulic diameter of the square channel), Here,

[0148]

number

[0149] (Pressure difference between the inlet and outlet)

[0150]

number

[0151] L = channel length, ρ = specific gravity of the liquid. The average shear stress over a given period is proportional to the following parameters:

[0152]

number

[0153] In a previous study using an experimental setup with h=220μm, b=400μm, θ=7° (OrganoPlate, Mimetas), and a tilt frequency of 16 mins, the maximum shear stress was estimated to be 1.7 dyne / cm² based on a numerical model simulated with Python software. The current configuration (h=400μm, b=1mm, θ=4°, tilt frequency of 1 min) yields 3.4 dyne / cm² at higher frequencies. 2 It was predicted that this would generate the maximum shear stress.

[0154] Induction of in vitro ischemic conditions To induce ischemia, the chips were placed in the incubation chamber of an EVOS fl automated imaging system containing 2% O2 and 5% CO2 for 24 hours. Before incubating the chips in a hypoxic chamber, the culture medium was flushed with nitrogen gas for 1 minute and replaced with serum and glucose-free DMEM (Gibco, catalog no.: 11966025) that had been stored overnight under the same hypoxic conditions (2% O2 and 5% CO2) before use. There was no flow during the ischemic period. Samples under normal oxygen conditions were cultured in an incubator with 5% CO2 and atmospheric O2 concentration (approximately 20%).

[0155] Measurement of cell viability and cytotoxicity Cell viability and cytotoxicity were measured using the Live / Dead® Viability kit (ThermoFisher Scientific, catalog number: L3224) and the LDH-Cytox Assay® kit (BioLegend, catalog number: 426401), respectively. Cell viability was calculated by dividing the number of viable cells by the total number of cells. Relative cytotoxicity of ischemia was calculated based on optical density (OD) read at a wavelength of 490 nm as follows:

[0156]

number

[0157] Functional characterization of the reconstructed BBB Evaluation of BBB as a physically intact barrier To evaluate the physical integrity of the blood-brain barrier (BBB) ​​formed on the microfluidic chip, FITC-conjugated dextran (70 kDa and 4 kDa) was injected into the "blood-side" channel, and diffusion across it was monitored. Fluorescence images (at 488 nm) were obtained at different time points 1 hour after probe injection, and fluorescence intensity was measured using ImageJ (NIH). The transmission coefficient was calculated using the following formula.

[0158]

number

[0159] A = surface area of ​​the membrane, C0 = initial concentration on the donor side.

[0160]

number

[0161] V brain = Hydrogel volume of "brain" channels,

[0162]

number

[0163]

number

[0164] I o = Initial fluorescence intensity This formula assumes that flux across the imaging boundary is negligible and that transendothelial flux is constant. In the tips described herein, these assumptions were met without issue at time intervals (Δt) of less than 15 minutes; i.e., P calculated for Δt of 5, 10 and 15 minutes. app There was no significant difference between them (n=5, p-value>0.05). Δt was set to 10 minutes. The following formula was used to analyze the entire barrier.

[0165]

number

[0166] and the endothelium itself

[0167]

number

[0168] Based on BBB

[0169]

number

[0170] I calculated it.

[0171]

number

[0172] Evaluation of the blood-brain barrier (BBB) ​​as a biochemically intact barrier. We investigated whether the formed endothelium could isolate neurons in the "brain" channel from the serum in the "blood side" channel. First, serum-containing medium with ECM and PM (9:1, v / v) containing 10% FBS was added to the "blood side" channel. After 24 hours of incubation, the behavior of microglia was studied by immunostaining with its reactivity marker, ionized calcium-binding adapter molecule 1 (IBA-1), and its activation marker, differentiation 68 (CD68).

[0173] Evaluation of the blood-brain barrier (BBB) ​​as a cell-selective barrier against invading cells We investigated whether the reconstructed BBB could exhibit different responses to various types of invading cells; two human breast cancer cell lines (MB-231 and its brain metastatic derivative population MB-231Br) were tested for this purpose. Cells were pre-stained with Vybrant® DiO cell labeling solution (Invitrogen, catalog number: V22886). For pre-staining, cells were incubated in a cell culture incubator with staining medium (5 μL of labeling solution per 1 mL of culture medium) for 20 minutes and washed three times with sterile PBS (phosphate-buffered saline, pH=7.4). Then, 10 μL of cells were divided into 1 × 10⁶ cells. 6 The cells were injected into the "blood side" channel at a density of cells / mL. The tip was slightly tilted to allow the cells to accumulate on the hydrogel boundary. Images were taken 1 or 10 days after cancer cell injection using the EVOS fl automated imaging system (Life Technologies).

[0174] Neurodegenerative staining Ischemia-induced neurodegeneration was studied using the Fluoro-Jade C (FJC) Staining Kit, following the manufacturer's protocol (Biosensis, biosensis® Ready-to-Dilute™ Fluoro-Jade® C Staining Kit, catalog number: TR-100-FJ) with some modifications. The incubation time for the staining solution was tripled in the protocol to ensure sufficient diffusion of the staining solution into the hydrogel of the central channel of the chip where neurons were growing. After staining, the samples were washed at least three times with PBS. The incubation time for each wash was 5–10 minutes. Images were acquired using an EVOS fl microscope (Life Technologies).

[0175] Immunocytochemistry (ICC) of NVU chips To fix the cells in the NVU chip, 50–60 μL of 4% paraformaldehyde (PFA) was added as a droplet to the inlet of each channel and held within the channel for at least 30 minutes. The fixed hydrogel matrix was gently washed by adding 30–40 μL of PBS droplet to the inlet of each channel. This washing was repeated at least 5 times. The cells in the NVU chip were then permeabilized with 0.1% Triton X-100 in PBS for 10–15 minutes. The permeabilized cells were washed 5 times with PBS and then blocked with 5% normal donkey serum in PBST (0.05% Tween20 in PBS) for 40 minutes to 1 hour. The blocked cells were incubated with primary antibody (30–40 μL per channel) at 4°C for at least one night.The dilutions of the primary antibodies were as follows: sheep polyclonal anti-human CD31 / PECAM-1 (R&D Systems, catalog number: AF806, 1:20), rabbit polyclonal anti-human GFAP (Sigma, catalog number: G9269, 1:100), chicken polyclonal anti-human GFAP (Synaptic Systems, catalog number: 173006, 1:500), rabbit polyclonal anti-human AQP4 (Novus Biologicals, Catalog No.: NBP1-87679, 1:2000), Mouse monoclonal anti-human ZO-1 (Invitrogen, Catalog No.: 339100, 1:100), Rabbit polyclonal anti-human von Willebrand factor (vWF, Sigma, Catalog No.: F3520, 1:200), Mouse monoclonal anti-human vWF (Sigma, Catalog No.: AMAB90931, 1:500), Mouse monoclonal anti-human podoplanin (PDPN) (E-1) (Santa Cruz Biotechnology, Catalog No.: SC376695, 1:100), Rabbit polyclonal anti-human synapsin 1 / 2 (Synaptic System, catalog number: 106003, 1:1,000), chicken polyclonal anti-human MAP2 (Abcam, catalog number: ab5392, 1:10,000), goat polyclonal anti-human IBA-1 (Abcam, catalog number: ab5076, 1:200), rabbit polyclonal anti-human IL-1β (Abcam, catalog number: ab9722, 1:100), mouse monoclonal anti-human CD68 (Bio-Rad, catalog number: MAC5709, 1:100), rabbit monoclonal anti-human CD44 (Invitrogen, catalog number: 19H8L4, 1:500), mouse monoclonal anti-human CD34 (Life (technology, catalog number: BI-3C5, 1:250), mouse monoclonal anti-human nestin (ThermoFisher, catalog number: MA1-5840, 1:250), rabbit monoclonal anti-human PDGFRβ (Cell Signaling, catalog number: 3169, 1:100), and mouse monoclonal anti-human HIF-1α (Abcam, catalog number: ab6066, 1:200).To prevent drying during primary antibody incubation, plates containing the tips were humidified with distilled water. Incubated samples were washed five times with blocking solution, and then various secondary antibodies (Jackson ImmunoResearch Laboratories), including DyLight405 anti-rabbit, Alexa Fluor488 anti-chicken, mouse, or rabbit, Alexa Fluor594 anti-mouse, rabbit, or sheep, and Alexa Fluor647 anti-mouse, rabbit, or sheep, were added to the samples at a 1:500 dilution at room temperature for at least 2 hours. Immunostained slides were mounted with ProLong Diamond anti-fade reagent (ThermoFisher) and cured for 24 hours. To prevent collapse of the fixed hydrogel structure, all buffers, including PFA, PBS, and blocking solution, were not completely removed from the outlet reservoir throughout the entire ICC procedure.

[0176] 3D image of BBB Fluorescence images of immunostained blood-brain barrier (BBB) ​​structures were acquired at 10x and 20x magnification using a Zeiss LSM 880 confocal microscope. NVU tips were scanned at different focal planes in the Z range of 0–100 μm, with intervals of 8–10 μm. A 3D viewer plugin for ImageJ was used for 3D reconstruction of the images.

[0177] Calcium imaging and analysis To record cytoplasmic calcium oscillations in neurons differentiated from iPSC-derived NPCs, iPSC-derived NPCs were pre-stained with DiL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Molecular probes, catalog no.: D282) before integration with other cells, as described in the process of brain tissue and BBB reconstruction. One hour prior to calcium imaging, 5 μM Fluo-4AM (Thermo Fisher Scientific, catalog no.: F14201) was added to the tip inlet. Spontaneous calcium oscillations were observed in DiL-labeled cells using a confocal microscope (Carl Zeiss, LSM 710, Gottingen, Germany) at 37°C and 5% CO2. Calcium signaling was recorded in time-lapse video recording mode for 10 minutes at a rate of 2 seconds / frame. Recorded images were analyzed using ImageJ software with the Time Series analyzer V3 plugin. Calcium dynamics in each DiL-labeled cell were tracked using ROI (region of interest) analysis, and parameters of calcium oscillations in each cell, such as frequency and amplitude (ΔF / F0), were calculated using a custom script written in IDL (Interactive Digital Language). The tracked calcium signal was subtracted from the extracellular background signal and then normalized to the intracellular baseline (F0).

[0178] Real-time quantitative PCR The gene expression patterns of the entire cell population were evaluated. RNA was extracted for analysis using more than six chips from each experimental condition. Cells from different batches were used to ensure a sufficient number of cells to prepare multiple chips simultaneously. The culture medium was removed from both side channels and replaced with fresh PBS. The process was repeated twice, with a 5-minute incubation in between. After PBS removal, 50 μL of RLT buffer + lysis buffer (Qiagen, catalog number: 1053393) was injected into both side channels and incubated for 5 minutes. Thorough pipetting was required to collect all cell types in the chip, especially those in the "brain" channel. Total RNA was then extracted using the RNeasy Mini Kit (Qiagen, catalog number: 74104) according to the manufacturer's protocol. RNA quality and concentration were determined by an Agilent 210 Bioanalyzer. The amount of RNA obtained from each chip was as follows for each experimental condition: a chip under normal oxygen conditions for 10 days produced approximately 250 ng; a chip with ischemia under the same conditions followed by 24 hours produced approximately 130 ng; a chip used as a control group for evaluating nerve repair effects under normal oxygen conditions for 18 days produced approximately 400 ng; a chip with reperfusion only produced approximately 200 ng; and a chip with transplanted stem cells produced approximately 350 ng to 750 ng, depending on the stem cell type. Total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, catalog number: 4368814). Real-time qPCR was performed on a StepOnePlus real-time PCR system (Applied Biosystems) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, catalog number: 1725272A) to quantify the expression level of the target gene. qPCR amplification was achieved over 40 cycles at 95°C for 30 seconds, 95°C for 15 seconds, and 65°C for 50 seconds. StepOnePlus real-time PCR software was used to distinguish the signal from noise, and further manual checks were performed to confirm that the obtained Ct values ​​were indeed derived from the genuine signal.Customized qPCR plates were designed and manufactured by Sciencell. Neurodevelopmental qPCR plates were purchased from Qiagen. Twelve genes with known ischemic behavior were initially selected, and the reproducibility of their expression was confirmed in triplicate. The expression of the final 123 genes was measured on more than six independent chips for each experimental condition.

[0179] Evaluation of the nerve repair capacity of stem cells The nerve repair capabilities of various types of stem cells were investigated. 24 hours after ischemic injury, serum and glucose-free DMEM medium was replaced in the "CSF side" channel with NDM / AM (a mixed medium of NDM, serum-free AM, and ACM in a ratio of 8:4:1 (v / v / v)). Stem cells were then placed in ECM / PM (a mixed medium of serum-containing ECM and PM in a ratio of 9:1 (v / v)) at a rate of 5 × 10⁶. 6 Collect at a density of cells / mL, 10 4 Cells were injected into the "blood side" channel. This setup corresponds to the intravascular route for stem cell transplantation, which is the most widely used route for stem cell therapy. Stem cells were incubated for 3 hours to allow cell adhesion, and suspended cells were gently washed with fresh ECM / PM. The culture medium in both side channels was changed daily for an additional 7 days before further analysis.

[0180] Stem cell tracking To track the behavior of stem cells on the chip, stem cells hNPC, hNSC(NR1), hAMSC, hBMSC, and hEPC were transfected using a lentiviral vector carrying green fluorescent protein (GFP). Due to insufficient viral transfection efficiency for hHSC, a pre-staining method was used for hHSC. Ready-to-use GFP lentiviral particles were purchased from GenTarget Inc (San Diego, CA, USA) and used according to the manufacturer's protocol with some modifications. More specifically, cells were cultured in 48-well plates until confluence reached 50%–75%. Before transduction, the cell culture medium was removed, and 0.25 mL of fresh medium and 15 μL of virus solution were added to each well. To achieve the desired transduction efficiency, cells were cultured in a cell culture incubator for 2–3 days without medium changes. hHSC was pre-stained with Vybrant® DiO cell labeling solution (Invitrogen, catalog number: V22886). 1 x 10 6 hHSCs at a concentration of cells / mL were incubated for 20 minutes in a cell culture incubator with staining medium (10 μL of labeled solution per 1 mL of culture medium) in a 96-well plate, and washed three times with sterile PBS (phosphate-buffered saline, pH=7.4) for use. After ischemic injury, each stem cell was injected into the "blood side" channel of the tip, and images were taken every other day for up to 7 days to track stem cell infiltration. At the end of the 7 days, stem cells were immunostained for stem cell markers (nestin for hNPCs and hNSCs, CD44 for hBMSCs and hAMSCs, and CD34 for hEPCs and hHSCs) and differentiation markers (MAP2 for neurogenesis, GFAP for glialogenesis, and von Willebrand factor for angiogenesis). The extent of extravasation of both cancer cells and stem cells was quantified by image scoring (ImageJ, NIH).

[0181] statistical methods Each independent experiment was repeated at least three times, and the results were presented as mean ± standard deviation (SD). For quantitative analysis of fluorescence images, at least three images were acquired from different samples, and the desired aspect was quantitatively analyzed using the image analysis software ImageJ (NIH). Statistical significance was assessed using a one-sided Student's t-test for comparisons between two groups, and a one-way ANOVA (Daniel's XL Toolbox) with a Bonferroni-Holm post-hoc test for comparisons between multiple groups. A p-value less than 0.05 was considered significant.

[0182] Further embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications, including U.S. Provisional Patent Application No. 63 / 013,903, which are mentioned herein and / or listed in the application datasheet, are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified to use the concepts of various patents, applications, and publications in order to provide further embodiments as needed.

[0183] These and other modifications may be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but rather as encompassing all possible embodiments along with the entire scope of equivalents for which such claims are entitled. Thus, the claims are not limited by this disclosure.

Claims

1. A biomimetic system, The biomimetic system comprises a microfluidic chip, and the microfluidic chip is plane; A first channel formed on the plane and having a first volume defined by a first width, a first height, and a first length, wherein the first volume extends in a first direction; and A second channel formed on the plane adjacent to the first channel, having a second volume defined by a second width, a second height, and a second length, wherein the second volume extends in the direction of the first channel and the second height is greater than the height of the first channel. Equipped with, The first channel is in fluid communication with the second channel through a first opening that extends along at least a portion of the first length, the first opening extending from the plane to the first height; The first and second heights are sized to generate sufficient surface tension at the first opening so that the liquid injected into the first channel or the second channel is substantially confined within the first volume or the second volume, respectively, or so that the flow of the liquid between them is controlled, and the surface tension generates a non-physical microfluidic barrier that restricts or selectively controls the passage of the liquid, and the biomimetic system An extracellular matrix confined within the first volume of the first channel, wherein the sidewall of the extracellular matrix extends across the first opening, forming the non-physical microfluidic barrier between the first channel and the second channel; and Epithelial barrier disposed at the first opening between the first channel and the second channel A biomimetic system that further incorporates these features.

2. A third channel formed on the plane adjacent to the first channel, having a third volume defined by a third width, a third height, and a third length, wherein the third volume extends in the direction of the first channel and the third height is greater than the height of the first channel. It further includes, The third channel is in fluid communication with the first channel through a second opening that extends along at least a portion of the first length, the second opening extending from the plane to the first height; The first and third heights are sized to generate sufficient surface tension at the second opening so that the liquid injected into the first or third channel is substantially confined within the first or third volume, respectively, or so that the flow of the liquid between them is controlled, and the surface tension generates a second non-physical microfluidic barrier that restricts or selectively controls the passage of the liquid. The biomimetic system according to claim 1.

3. A third channel formed on the plane adjacent to the second channel, having a third volume defined by a third width, a third height, and a third length, wherein the third volume extends in the first direction and the third height is lower than the second height. It further includes, The third channel is in fluid communication with the second channel through a second opening that extends along at least a portion of the second length, the second opening extending from the plane to the third height; The second and third heights are sized to generate sufficient surface tension at the second opening so that the liquid injected into the second or third channel is substantially confined within the second or third volume, respectively, or so that the flow of the liquid between them is controlled, thereby creating a second non-physical microfluidic barrier that restricts or selectively controls the passage of the liquid. The biomimetic system according to claim 1.

4. The biomimetic system according to any one of claims 1 to 3, wherein the epithelial barrier is continuous.

5. The biomimetic system according to any one of claims 1 to 4, wherein the epithelial barrier includes endothelial cells.

6. The biomimetic system according to claim 5, wherein the epithelial barrier is an endothelial barrier.

7. The biomimetic system according to claim 6, wherein the endothelial barrier comprises brain microvascular endothelial cells (BMECs).

8. The biomimetic system according to claim 6, wherein the endothelial barrier includes human BMEC.

9. The biomimetic system according to any one of claims 1 to 8, further comprising pericytes in the second channel.

10. The biomimetic system according to any one of claims 1 to 9, further comprising a first cell in the extracellular matrix of the first channel.

11. The biomimetic system according to claim 10, wherein the first cell is a nerve cell.

12. The biomimetic system according to claim 11, wherein the nerve cells include neural progenitor cells, astrocytes, microglia, or a combination thereof, derived from human induced pluripotent stem cells.

13. The biomimetic system according to claim 10, wherein the first cell is a cardiac cell, skeletal muscle cell, hepatic cell, renal cell, osteocyte, skin cell, esophageal cell, intestinal cell, gastric cell, colon cell, lung cell, or pancreatic cell.

14. The biomimetic system according to any one of claims 1 to 13, wherein the extracellular matrix comprises a hydrogel.

15. The biomimetic system according to claim 14, wherein the hydrogel comprises a basement membrane extract (BME).

16. The biomimetic system according to any one of claims 1 to 15, further comprising a first medium confined within the second volume of the second channel.

17. The biomimetic system according to any one of claims 2 to 16, wherein the third height is greater than the first height, and the biomimetic system further includes a second medium confined within the third volume of the third channel.

18. The biomimetic system according to any one of claims 2 to 16, wherein the third height is lower than the second height, and the biomimetic system further comprises a second extracellular matrix confined within the third volume of the third channel.

19. A biomimetic system according to any one of claims 1 to 18, wherein the tissue model has a vascular peduncle.

20. The biomimetic system according to claim 19, wherein the tissue model with a vascular peduncle is a blood-brain barrier model or a stroke model.

21. The biomimetic system according to claim 19, wherein the tissue model with a vascular peduncles is a heart model, a skeletal muscle model, a liver model, a kidney model, a bone model, a skin model, an esophageal model, a stomach model, a colon model, an intestine model, a lung model, or a pancreas model.

22. A method for preparing a biomimetic system according to any one of claims 1 to 21, Depositing an extracellular matrix precursor in the first channel of the microfluidic chip; and The extracellular matrix precursor is cured to deliver the extracellular matrix to the first channel. Methods that include...

23. The method according to claim 22, wherein curing the extracellular matrix precursor comprises incubating the extracellular matrix precursor, and the method further comprises depositing the first medium in the second channel adjacent to the extracellular matrix of the first channel.

24. The method according to claim 22 or 23, further comprising culturing a continuous endothelial barrier in the first opening between the first channel and the second channel, wherein the first medium further comprises pericytes.

25. A method for screening therapeutic agents, Depositing the therapeutic agent in the second channel of the microfluidic chip of the biomimetic system according to any one of claims 1 to 21; and To image the aforementioned microfluidic chip Methods that include...

26. The method according to claim 25, wherein the therapeutic agent comprises stem cells, small molecules, or peptides.

27. The method according to claim 25 or 26, further comprising carrying out the method according to any one of claims 22 to 24.

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