Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
BLOOD BRAIN BARRIER MODEL IN A 3D CO-CULTURE MICROFLUIDIC SYSTEM
Document Type and Number:
WIPO Patent Application WO/2017/035119
Kind Code:
A1
Abstract:
An efficient in-vitro Blood Brain Barrier (BBB) model consisting of the key cells of the BBB - namely, neurons, astrocytes and endothelial cells - is provided, which permits recapitulating the in-vivo BBB and shedding light on contributions from each individual cell type. A microfluidic system for modeling the blood brain barrier comprises an optically transparent substrate, the substrate comprising: (i) at least one fluid channel; (ii) a first gel channel comprising a first gel region; (iii) a second gel channel comprising a second gel region; and (iv) at least one row of posts.

Inventors:
KAMM ROGER DALE (US)
MA DONG LIANG (SG)
GOH EYLEEN LAY KEOW (SG)
ADRIANI GIULIA (SG)
PAVESI ANDREA (SG)
Application Number:
PCT/US2016/048138
Publication Date:
March 02, 2017
Filing Date:
August 23, 2016
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
NAT UNIV SINGAPORE (SG)
MASSACHUSETTS INST TECHNOLOGY (US)
International Classes:
G01N33/48; B01L3/00; C12M1/00; C12M3/00; C12N5/079
Domestic Patent References:
WO2012050981A12012-04-19
Foreign References:
CN104630059A2015-05-20
US8815584B12014-08-26
US20140065660A12014-03-06
US20110104658A12011-05-05
US20120211373A12012-08-23
US20140142370A12014-05-22
Other References:
WOLFF, A ET AL.: "In vitro blood-brain barrier models-An overview of established models and new microfluidic approaches.", JOURNAL OF PHARMACEUTICAL SCIENCES., vol. 104, no. 9, January 2015 (2015-01-01), pages 2728, 2731, XP055367090
WITTIG JR, JH ET AL.: "A reusable microfluidic plate with alternate-choice architecture for assessing growth preference in tissue culture.", JOURNAL OF NEUROSCIENCE METHODS., vol. 144, no. 1, 2005, XP027670256
BHATIA, SN ET AL.: "Microfluidic organs-on-chips.", NATURE BIOTECHNOLOGY, vol. 32, no. 8, 2014, pages 760 - 772, XP002761628
Attorney, Agent or Firm:
CARROLL, Alice, O. et al. (US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. A microfluidic system for modeling the blood brain barrier, the microfluidic system comprising:

an optically transparent substrate, the substrate comprising

(i) at least one fluid channel;

(ii) a first gel channel comprising a first gel region;

(iii) a second gel channel comprising a second gel region;

(iv) at least one row of posts, at least a first row of posts of the at least one row of posts confining the first gel region, and at least a second row of posts of the at least one row of posts confining the second gel region;

at least a portion of the first gel region flanking at least a portion of a first fluid channel of the at least one fluid channel; and

at least a portion of the second gel region flanking at least a portion of a second fluid channel of the at least one fluid channel.

2. The system of Claim 1, further comprising a co-culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron.

3. The system of Claim 2, further comprising:

the at least one endothelial cell and endothelial cell culture media in the first fluid channel;

a solution comprising a first biologically relevant gel and the at least one astrocyte in the first gel region;

a solution comprising a second biologically relevant gel and the at least one neuron in the second gel region; and

culture media for neurons in the second fluid channel.

4. The system of Claim 3, wherein at least one of the first biologically relevant gel and the second biologically relevant gel comprises a hydrogel solution comprising collagen.

5. The system of Claim 1, wherein the first row of posts and the second row of posts are parallel, the first fluid channel flanking the first gel region through the first row of posts, and the second gel region flanking the second fluid channel through the second row of posts.

6. The system of Claim 5, further comprising a third row of posts of the at least one row of posts;

at least a portion of each of the first fluid channel, the first gel region, the second gel region and the second fluid channel being mutually parallel to each other along at least a portion of the microfluidic system;

the first gel region flanking the second gel region through the third row of posts.

7. The system of Claim 1, wherein each post of the at least one row of posts forms a triangular shape, a trapezoidal shape or a combination thereof.

8. The system of Claim 1, wherein a distance between each neighboring pair of posts in the at least one row of posts is from about 50 micrometers to about 300 micrometers.

9. The system of Claim 1, wherein a height of each of the at least one fluid channel, the first gel channel and the second gel channel is between about 50 micrometers and about 200 micrometers.

10. The system of Claim 1, wherein a width of at least one of the first gel region and the second gel region is between about 200 microns and about 1000 microns.

11. The system of Claim 1, further comprising a co-culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron, and wherein the at least one astrocyte and the at least one neuron are cultured as a plurality of such cells extending to a substantial extent in each of three spatial dimensions.

12. The system of Claim 1, further comprising a co-culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron; the at least one endothelial cell cultured as a plurality of endothelial cells to form a monolayer in the first fluid channel, the at least one astrocyte exhibiting a star- shaped morphology in the first gel region; and the at least one neuron exhibiting neurite outgrowth in the second gel region.

13. The system of Claim 1, further comprising a co-culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron, wherein each cell type of the at least one endothelial cell, the at least one astrocyte and the at least one neuron exhibits cell growth, expresses cellular markers and displays morphological characteristics specific to its cell type.

14. The system of Claim 1, further comprising at least one neuron comprising at least two neuron branch points, in the second gel region.

15. The system of Claim 1, further comprising at least two neurons comprising synaptic connectivity between the at least two neurons.

16. The system of Claim 1, further comprising a co-culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron; and comprising a stain permitting visualization of at least one of the at least one endothelial cell, the at least one astrocyte and the at least one neuron through the optically transparent substrate.

17. The system of Claim 1, further comprising a monolayer of endothelial cells cultured in the microfluidic system.

18. The system of Claim 17, wherein the monolayer of endothelial cells extends along at least a portion of an interface between the first fluid channel and the first gel region.

19. The system of Claim 17, wherein the monolayer of endothelial cells is selectively permeable to at least one tracer compound based on the size of the at least one tracer compound.

20. A microfluidic system for modeling the blood brain barrier, the microfluidic system comprising:

an optically transparent substrate, the substrate comprising

(i) at least one fluid channel;

(ii) at least one gel channel;

(iii) at least one row of posts confining at least one gel region of a gel channel of the at least one gel channels; a co-culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron.

The system of Claim 20, further comprising:

a solution comprising a biologically relevant gel, the at least one astrocyte and the at least one neuron, in the at least one gel region of the gel channel of the at least one gel channels.

The system of Claim 21, further comprising a solution comprising a second biologically relevant gel and no cells, in another gel region of another gel channel of the at least one gel channels.

The system of Claim 20, further comprising at least one type of culture media in the at least one fluid channel, and: (i) a first solution comprising a first biologically relevant gel, at least one first astrocyte of the at least one astrocyte, and at least one first neuron of the at least one neuron, in a first gel region of the at least one gel regions; and (ii) a second solution comprising a second biologically relevant gel, at least one second astrocyte of the at least one astrocyte and at least one second neuron of the at least one neuron, in a second gel region of the at least one gel regions.

Description:
BLOOD BRAIN BARRIER MODEL IN A 3D CO-CULTURE MICROFLUIDIC SYSTEM

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.

62/209,013, filed on August 24, 2015. The entire teachings of the above application(s) are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The Blood Brain Barrier (BBB) is a selective barrier that restricts compounds entering the central nervous system (CNS). This tight regulation is important for maintaining homeostasis of the neural microenvironment and protecting the CNS from chemical insults and damage [1]. However, this protective barrier may also hinder drug delivery. The in-vivo BBB consists of brain endothelial cells (EC), astrocytes, pericytes, smooth muscle cells, and glial cells. Endothelial cells form the wall of capillaries, and astrocytes form a complex network surrounding the capillaries. This close cell association between neurons, astrocytes and endothelial cells is important in induction and maintenance of the barrier properties [1].

[0003] Microfluidic systems have been successfully used to carry out wide-ranging experiments such as angiogenesis [2,3], cancer cell intravasation [4] and drug screening [5]. However, most of the current BBB models are two-dimensional (2-D) culture systems that consist of endothelial cells in monoculture [6], [7], in co-culture with glial cells or astrocytes- conditioned media [8]-[l l] and neurons [12], [13]. Similar culture conditions have been investigated in 2-D models developed within microfluidic platforms [14], [15]. Moreover, in a previous work, fluid flow in the microfluidic platforms was considered to investigate the effect of shear stress on endothelial junction formation [16]. These existing models may recapitulate some of the major BBB functions, but still fail to address the three-dimensional (3D) cellular organization that remains crucial for cellular processes in-vivo. A recent study showed a 3D multicellular spheroid system consisting of human primary brain endothelial cells, primary pericytes and primary astrocytes spontaneously self-organizing into a defined multicellular BBB structure [17]. Although these studies demonstrated the importance of physiologically relevant culture conditions for in-vitro models, these existing BBB models do not allow specific manipulation and monitoring of BBB development for drug delivery studies. Moreover, there is currently no in-vitro 3D BBB microfluidic model available that allows studies on drug screening through the BBB and on neuronal growth on one single platform.

SUMMARY OF THE INVENTION

[0004] In accordance with a version of the invention, an efficient in-vitro Blood Brain Barrier (BBB) model consisting of the key cells of the BBB - namely, neurons, astrocytes and endothelial cells (EC) - is provided, which permits recapitulating the in-vivo BBB and shedding light on contributions from each individual cell type.

[0005] In one version according to the invention, there is provided a microfluidic system for modeling the blood brain barrier. The microfluidic system comprises an optically transparent substrate, the substrate comprising: (i) at least one fluid channel; (ii) a first gel channel comprising a first gel region; (iii) a second gel channel comprising a second gel region; and (iv) at least one row of posts. At least a first row of posts of the at least one row of posts confines the first gel region, and at least a second row of posts of the at least one row of posts confines the second gel region. At least a portion of the first gel region flanks at least a portion of a first fluid channel of the at least one fluid channel; and at least a portion of the second gel region flanks at least a portion of a second fluid channel of the at least one fluid channel.

[0006] In further, related versions of the invention, the system may further comprise a co- culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron. The system may further comprise the at least one endothelial cell and endothelial cell culture media in the first fluid channel; a solution comprising a first biologically relevant gel and the at least one astrocyte in the first gel region; a solution comprising a second biologically relevant gel and the at least one neuron in the second gel region; and culture media for neurons in the second fluid channel. At least one of the first biologically relevant gel and the second biologically relevant gel may, for example, comprise a hydrogel solution comprising collagen. The first row of posts and the second row of posts may be parallel, the first fluid channel flanking the first gel region through the first row of posts, and the second gel region flanking the second fluid channel through the second row of posts. The system may further comprise a third row of posts of the at least one row of posts; at least a portion of each of the first fluid channel, the first gel region, the second gel region and the second fluid channel being mutually parallel to each other along at least a portion of the microfluidic system; and the first gel region flanking the second gel region through the third row of posts.

[0007] In other related versions of the invention, each post of the at least one row of posts may form a triangular shape, a trapezoidal shape or a combination thereof. A distance between each neighboring pair of posts in the at least one row of posts may be from about 50 micrometers to about 300 micrometers. A height of each of the at least one fluid channel, the first gel channel and the second gel channel may be between about 50 micrometers and about 200 micrometers. A width of at least one of the first gel region and the second gel region may be between about 200 microns and about 1000 microns.

[0008] In further, related versions of the invention, the system may further comprise a co- culture in the microfluidic system, the co-culture including at least one endothelial cell, at least one astrocyte and at least one neuron, wherein the at least one astrocyte and the at least one neuron are cultured as a plurality of such cells extending to a substantial extent in each of three spatial dimensions. The at least one endothelial cell may be cultured as a plurality of endothelial cells to form a monolayer in the first fluid channel, the at least one astrocyte exhibiting a star-shaped morphology in the first gel region, and the at least one neuron exhibiting neurite outgrowth in the second gel region. Each cell type of the at least one endothelial cell, the at least one astrocyte and the at least one neuron may exhibit cell growth, express cellular markers and display morphological characteristics specific to its cell type. The system may further comprise at least one neuron comprising at least two neuron branch points, in the second gel region; and may further comprise at least two neurons comprising synaptic connectivity between the at least two neurons. The system may further comprise a stain permitting visualization of at least one of the at least one endothelial cell, the at least one astrocyte and the at least one neuron through the optically transparent substrate. The system may further comprise a monolayer of endothelial cells cultured in the microfluidic system. The monolayer of endothelial cells may extend along at least a portion of an interface between the first fluid channel and the first gel region; and may be selectively permeable to at least one tracer compound based on the size of the at least one tracer compound.

[0009] In another version of the invention, there is provided a microfluidic system for modeling the blood brain barrier. The microfluidic system comprises an optically transparent substrate, the substrate comprising: (i) at least one fluid channel; (ii) at least one gel channel; and (iii) at least one row of posts confining at least one gel region of a gel channel of the at least one gel channels. The system comprises a co-culture in the microfluidic system, the co- culture including at least one endothelial cell, at least one astrocyte and at least one neuron.

[0010] In further, related embodiments, the system may further comprise a solution comprising a biologically relevant gel, the at least one astrocyte and the at least one neuron, in the at least one gel region of the gel channel of the at least one gel channels. The system may further comprise a solution comprising a second biologically relevant gel and no cells, in another gel region of another gel channel of the at least one gel channels.

[0011] In another related embodiment, the system may further comprise at least one type of culture media in the at least one fluid channel, and: (i) a first solution comprising a first biologically relevant gel, at least one first astrocyte of the at least one astrocyte, and at least one first neuron of the at least one neuron, in a first gel region of the at least one gel regions; and (ii) a second solution comprising a second biologically relevant gel, at least one second astrocyte of the at least one astrocyte and at least one second neuron of the at least one neuron, in a second gel region of the at least one gel regions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0013] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

[0014] FIG. 1 A is a schematic layout of a 3D PDMS microfluidic device in accordance with a version of the invention, and an enlarged view of the channels in the device. Two central gel regions for co-culturing astrocytes (in blue) and neurons (in orange), are shown; and two side channels for hosting endothelial cells and media (in green and red, respectively), are shown.

[0015] FIG. IB is a time line of an experiment, in accordance with a version of the invention. [0016] FIG. 1C is a set of phase contrast images showing growth of endothelial cells (HUVECs and hCMEC/D3), primary astrocytes and primary neurons in allocated

microfluidic channels, in an experiment in accordance with a version of the invention. Scale bars are 100 μιη.

[0017] FIGS. 2A-2C are a set of images showing the immunocytochemistry of primary neurons, primary astrocyte and endothelial cells with specific cell type markers, in

accordance with a version of the invention. FIG. 2A is a set of representative images showing top and side views of the three cell types in 3D co-culture, in accordance with a version of the invention. FIG. 2B is a 3D view of a neuron channel, in accordance with a version of the invention. FIG. 2C is a set of representative images showing immature neurons identified by DCX, astrocytes characterized by GFAP and HUVEC expressing VE- cadherin, in accordance with a version of the invention. The scale bar in FIGS. 2A and 2B is 200 μπι, and the scale bar in FIG. 2C is 50 μπι.

[0018] FIGS. 3A-3F are a set of images showing endothelial barrier characterization, in accordance with a version of the invention. FIGS. 3 A and 3B are representative images showing HUVEC (FIG. 3 A) and hCMEC/D3 (FIG. 3B) monolayers expressing F-actin and VE-cadherin. FIGS. 3C and 3D are three-dimensional visualizations, and FIGS. 3E and 3F are sections, of the endothelial walls for HUVEC (FIGS. 3C and 3E) and hCMEC/D3 (FIGS. 3D and 3F). Scale bar 50 μπι.

[0019] FIG. 3G is a graph showing calculated permeability coefficient of lOkDa and 70KDa dextrans comparing HUVEC and hCMEC/D3 at 4 DIV, in accordance with a version of the invention. FIG. 3H is a graph showing calculated permeability coefficient of lOkDa and 70KDa dextrans comparing hCMEC/D3 at 4 DIV and 7 DIV, in accordance with a version of the invention. FIG. 31 is a graph showing calculated permeability coefficient of lOkDa and 70KDa dextrans comparing HUVEC and hCMEC/D3 in the triple co-culture system, in accordance with a version of the invention. Data in FIGS. 3G-3I show mean values and SEM. Student's t-test results are shown, where the symbol "*" is for p<0.05, **for p<0.01, *** for pO.001, and **** for pO.0001.

[0020] FIGS. 4A-4E are images, graphs and plots showing a morphological assessment of primary neuron development in a 3D gel, in accordance with a version of the invention. FIG. 4A is a set of representative images showing neurons growing at 3, 7 and 11 days in- vitro (DIV). FIG. 4B is a set of representative images of neurite reconstruction, color-coded by branch level at 3, 7 and 11 DIV, with a scale bar of 100 μιη. FIG. 4C is a graph showing the sum of neurite lengths for each neuron at 3, 7 and 11 DIV; five regions of interest (ROI) were considered per each time point; the percentage of neurons for three ranges of neurite lengths are shown (0-700 μιη; 700-1400 μιη; 1400-2100 μιη). FIG. 4D is a plot of the number of segments per ROI for each branch level at 3, 7 and 11 DIV; the data show mean values and SEM; a statistical analysis using one-way ANOVA, is shown, where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FIG. 4E is a plot of the number of segments per neuron at 3, 7 and 11 DIV; the data show mean values and SEM; a statistical analysis using student's t-test, is shown, where ****p<0.0001.

[0021] FIGS. 5 A-5C are a set of images and graphs showing a functional characterization of neurons, in accordance with a version of the invention. FIG. 5A is a set of representative fluorescence and phase contrast images showing X-Rhod-1 (red) in neurons. FIG. 5B is a graph of representative fluorescence intensities from one neuron measured over the entire imaging time frame (250 s with images taken every 500 ms). FIG. 5C is a graph showing normalized fluorescence intensities for the neurons examined; the data show mean values and SEM.

DETAILED DESCRIPTION OF THE INVENTION

[0022] A description of example embodiments of the invention follows.

[0023] In accordance with a version of the invention, an efficient in-vitro Blood Brain Barrier (BBB) model consisting of the key cells of the BBB - namely, neurons, astrocytes and endothelial cells (EC) - is provided, which permits recapitulating the in-vivo BBB and shedding light on contributions from each individual cell type.

[0024] In one version according to the invention, a microfluidic device consists of two central gel regions flanked by two media channels, as described further relative to FIG.1A, below.

[0025] In an experiment in accordance with a version of the invention, described further in the Experimental section, below, rat primary neurons and astrocytes were used together with human endothelial cell lines (HUVECs and hCMEC/D3) to develop a three-dimensional (3D) in-vitro BBB model within a microfluidic device; but other cell types, such as those derived from induced pluripotent stem cells, could also be used. To achieve the 3D in-vitro BBB model, the walls of the brain vasculature were incorporated into the microfluidic device, by culturing endothelial cells in a triple co-culturing system with astrocytes and neurons.

[0026] FIG. 1 A is a schematic layout of a 3D PDMS microfluidic device 100 in accordance with a version of the invention, and an enlarged view of the channels in the device (inset). Two gel channels that include central gel regions 101, 102 for co-culturing astrocytes 101 (in blue) and neurons 102 (in orange), are shown; and two side fluid channels 103, 104 for hosting endothelial cells 103 and media 104 (in green and red, respectively), are shown. The system includes an optically transparent substrate 105, which may be made, for example, of poly(dimethylsiloxane) (PDMS). The fluid channels 103, 104 each include input/output ports (106, 107) and (108, 109); and the gel channels likewise include input/output ports (110, 111) and (112, 113). Three parallel rows of posts 114, 115 and 116 confine the gel regions 101, 102 and define the interface between flanking regions. The first fluid channel 103, for hosting endothelial cells, flanks the first gel region 101, which cultures astrocytes, through the first row of posts 114. The second gel region 102 flanks the second fluid channel 104, which hosts media for neurons, through the second row of posts 115. The first gel region 101 flanks the second gel region 102, which cultures neurons, through the third row of posts 116. The system 100 includes endothelial cells and endothelial cell culture media in the first fluid channel 103; a solution comprising a first biologically relevant gel and astrocytes in the first gel region 101; a solution comprising a second biologically relevant gel and neurons in the second gel region 102; and culture media for neurons in the second fluid channel 104. As used herein, the "biologically relevant gel" may, for example, be collagen, Matrigel ® (sold by Corning, Inc. of Corning, NY, U.S.A.), fibronectin, or hyaluronan; and may, in a particular example, be a hydrogel solution comprising collagen.

[0027] In one version according to the invention, each post rows of posts 114, 115 and 116 forms a triangular shape, a trapezoidal shape or a combination thereof. As used herein, a "triangular" or "trapezoidal" shape need not be an ideal triangle or trapezoid, but can, for example, include rounded corners or edges. A distance between each neighboring pair of posts in the at least one row of posts can, for example, be from about 50 micrometers to about 300 micrometers. A height of each of the at least one fluid channel 103, 104, the first gel channel and the second gel channel can, for example, be between about 50 micrometers and about 200 micrometers. A width of at least one of the first gel region 101 and the second gel region 102 can, for example, be between about 200 microns and about 1000 microns. Posts, gel regions, gel channels and fluid channels, and other aspects of microfluidic devices, may be used that are taught in U.S. Patent App. Pub. No. 2014/0057311 Al of Kamm et al, the entire teachings of which application are incorporated herein by reference.

[0028] As described further in the Experimental section, below, in accordance with a version of the invention, the microfluidic system 100 can include a co-culture of at least endothelial cells, astrocytes and neuron. Other cells, including other cells of the BBB, can be included. The astrocytes and neurons can be cultured to extend to a substantial extent in each of three spatial dimensions. In the co-culture, the endothelial cells can form a monolayer in the first fluid channel 103, the astrocytes can exhibit a star-shaped morphology in the first gel region 101; and the neurons can exhibit neurite outgrowth in the second gel region 102.

Furthermore, in the co-culture, each cell type of the endothelial cells, the astrocytes and the neurons can exhibit cell growth, express cellular markers and display morphological characteristics specific to its cell type. The neurons can form branches and exhibit synaptic connectivity. Staining can permit visualization of the endothelial cells, the astrocytes and the neurons through the optically transparent substrate. A monolayer of endothelial cells can be cultured in the microfluidic system, and can extend along at least a portion of an interface between the first fluid channel 103 and the first gel region 101. The monolayer of endothelial cells can be selectively permeable to at least one tracer compound based on the size of the at least one tracer compound.

[0029] Although FIG. 1 A is described as having astrocytes in a separate gel region 101 from neurons, which are in gel region 102, another version according to the invention includes a mixture of both astrocytes and neurons, either in the same gel region (such as either of gel regions 101 or 102), or in each of two or more gel regions (such as both of gel regions 101 and 102). In one such version, only a single gel region 101 is used, containing a mixture of both astrocytes and neurons. In that version, the system can have two rows of posts 114 and 116, with the second gel cage 102 and one of the rows of posts 115 being omitted, so that the second fluid channel 104 flanks directly onto row of posts 116 while the first fluid channel 103 flanks onto row of posts 114. In another such version, two or more gel regions 101 and 102 are used, as in FIG. 1 A, with at least one of the gel regions 101 and 102 containing a mixture of astrocytes and neurons. In such a case, one or more of the gel regions may be empty, while one or more other gel regions contain a mixture of astrocytes and neurons; or each of the gel regions may contain a mixture of astrocytes and neurons. [0030] FIGS. IB through 5C are described in further detail in the Experimental section, below, with reference to the Brief Description of the drawings, above.

[0031] Modeling the blood brain barrier (BBB) in a microfluidic platform in accordance with a version of the invention will offer the potential for high throughput screening, together with other advantages of microfluidic technologies, such as: as reduced quantity of media, cells and chemicals; lower costs; and a more precise control of the spatiotemporal parameters with respect to other conventional in-vitro and in-vivo assays.

[0032] Advantages of a BBB microfluidic model in accordance with a version of the invention can include:

[0033] 1. 3D microenvironment optimized for multi-cellular co-culture, e.g., endothelial cells, neurons and astrocytes growth;

[0034] 2. Precise control over spatiotemporal parameters;

[0035] 3. Real time visualization, in a fashion that is easier than in conventional systems, such as a Transwell® (trademark of Corning Life Sciences, Corning, NY, U.S.A.) system;

[0036] 4. Reduced volume of reagents and biological samples compared to standard culture dishes;

[0037] 5. Ability to include endothelial cells and astrocytes together with neurons. This allows a single platform for the study of BBB functions, the potential for high throughput screening for the permeability of drugs or compounds of interest and consequently, their ability to affect neuronal growth and functions.

[0038] Specific industrial applications of versions according to the invention include drug screening, and clinical screening for patient-specific therapy.

[0039] In accordance with a version of the invention, permeability can be improved, with a goal of reaching in-vivo values, by chemically stimulating the endothelial cells with compounds such as Ang-1 or cAMP to tighten the intercellular junctions, or by inclusion of pericytes in the culture.

[0040] In one version of the invention, the microfluidic system can include human induced pluripotent stem cells {e.g., provided by Cellular Dynamics International, Inc. of Madison, WI, U.S.A.) or patient-derived cells, for a personalized BBB model.

[0041] In addition, a more sophisticated in-vitro 3D BBB model can be provided by adding other cellular components of the BBB, such as pericytes and microglia, to a version of the invention. Experimental

1. Device Fabrication

[0042] The microti ui die device 100 (FIG. 1 A), in an experiment in accordance with a version of the invention, is a single layer device made of poly(dimethylsiloxane) (PDMS, Sylgard 184 Silicone elastomer kit, Dow Corning, Midland, MI, USA) by softlithography from a patterned SU-8 silicon wafer. Silicone elastomer and curing agent mixed at a weight ratio of 10: 1 were degassed, poured on the photolithographically patterned SU-8 structures and cured in the oven at 80 °C for 2 h. Input/output (I/O) ports 106, 107, 108, 109, 110, 111, 112 and 113 were created with biopsy punches and the device was sterilized by autoclave. Glass coverslips were plasma bonded to the PDMS layer to create closed channels of 150 μιη in height. This version of the device consists of four channels (FIG. 1 A), two for 3D hydrogels 101, 102 and two for culture media 103, 104 (FIG. 1A).

2. 3D Hydrogel Filling

[0043] The microchannels were coated with 1 mg/ml Poly-D-lysine (PDL) solution (Sigma- Aldrich, St. Louis, MO) to prevent the detachment of hydrogels from the channel walls (Shin et al. 2012). Two hydrogel solutions containing collagen type I (BD Biosciences, Franklin Lakes, NJ) were prepared with collagen concentrations of 7 mg/ml (for astrocytes) or 2.5 mg/ml (for neurons) at pH -7.4. Primary rat astrocytes or neurons were mixed in each of their specific hydrogel at the cell densities of 0.6 χ 10 6 cells/ml or 5 χ 10 6 cells/ml, respectively. The first hydrogel solution with astrocytes 101 was injected into the devices and allowed to polymerize in a C0 2 incubator (37 °C, 5 % C0 2 ) for 30 min followed by injection of the second hydrogel solution 102 with neurons and addition of supplemented-MEM into the lateral fluidic channel 103 close to astrocytes. After the second hydrogel polymerization in the C0 2 incubator (37 °C, 5 % C0 2 ) for 30 min, supplemented-MEM was injected into the lateral fluidic channel close to neurons 104. Media was changed to neurobasal medium ( B) supplemented with lx GlutaMAX-I and lx B27 (Life Technologies) after 24 h only in the fluidic channel 104 adjacent to hydrogel containing neurons. The two media were refreshed every 24 h for 7 days before seeding of endothelial cells.

3. Endothelial Cells Seeding

[0044] After 7 days in-vitro (DIV), the fluidic channels were incubated with collagen (100 μg/ml) in PBS for 45 min in a C0 2 incubator (37 °C, 5 % C0 2 ) to promote cell adhesion of endothelial cells. Two human endothelial cell lines were compared in the in-vitro BBB system, namely HUVECs isolated from umbilical vein and hCMEC/D3 isolated from cerebral microvessel. After coating, HUVECs (5 10 6 cells/ml) or hCMEC/D3 (8 10 6 cells/ml) in EGM-2 were seeded in the fluidic channel 103 adjacent to hydrogel 101 containing astrocytes. Non-adherent cells were removed 2h after seeding. Both supplemented neurobasal media and EGM-2 were refreshed every 24 h in the devices for 4 days (HUVECs) or 7 days (hCMEC/D3).

4. Morphological Analysis

a. Immunocytochemistry of Multiple Cell Types in Microfluidic

System

[0045] To characterize the multiple cell types in the 3D microfluidic devices,

immunocytochemistry was used to stain primary neurons, primary astrocyte and endothelial cells. Doublecortin (DCX) is a microtubule-associated protein specific for immature neurons, and antibody against DCX allows a clear visualization of the cell body and neurites.

Astrocytes were identified with an antibody against glial fibrillary acidic protein (GFAP). VE-cadherin expression showed the formation of intercellular junctions and the monolayer regularity of endothelial cells. The side and top views of the three types of cells in the 3D microfluidic devices showed neurons identified by DCX, astrocytes positive for GFAP and HUVEC expressing GFP (FIG. 2A). Neurons and astrocytes in 3D hydrogels showed specific cell morphologies that are signatures of both cell types: neurons showed neurite outgrowth (FIG. 2B and 2C) while astrocytes exhibited characteristic star-shaped morphology (FIG. 2C). HUVECs formed a monolayer in the channel after 4 days of culture in the device (FIG. 2C). These staining indicated that all the three different cell types were able to grow, express cellular markers and display morphological characteristics specific for each individual cell type.

b. Morphological Characteristics of the Endothelial Barrier

[0046] The integrity of the in-vitro monolayer formed by endothelial cells was assessed, to mimic the BBB in-vivo. Both HUVEC and hCMEC/D3 were able to form monolayers in the micro-channels in 4 and 7 days, respectively. Both cell types expressed F-actin and the intercellular junction protein VE-cadherin as shown in FIG. 3A and 3B. Representative 3D reconstructions (Fig. 3C and 3D) and cross sections (Fig. 3E and 3F) of the endothelial monolayers showed the integrity of the wall on the side of the channel at the interface between the medium channel 103 and the hydrogel containing astrocytes 101. c. Neuronal Filaments Analysis

[0047] For morphological analysis, the growth of these neurons was examined in the 3D hydrogel within the microfluidic device over time (FIG. 4). 3D reconstructed images (FIG. 4A) showed that the neurons were growing progressively in 3D within the hydrogel over the three time points examined (3, 7 and 11 DIV). Neurite growth was analyzed with the

IMARIS software (Bitplane, Zurich, Switzerland). Specifically, the imaging software defines a segment as the portion of a neurite between two branch points and the branch level as a number that increases moving outward. The branch level is determined by the diameter of the individual segments. The initial branch level is 1. At each branch point, the branch level of the segment with smaller mean diameter increases by one, while the segment with the greater diameter maintains the same branch level. The branch levels are plotted as color-coded images for three representative regions of interest (ROI) at 3, 7 and 11 DIV (FIG. 4B). The total neurite length and the percentage of neurons having lengths of 0-700 μπι; 700-1400 μπι; 1400-2100 μπι were calculated at 3, 7 and 11 DIV (FIG. 4). Additionally, the number of segments per ROI for each branch level at 3, 7 and 11 DIV (FIG. 4D) and the number of segments per neuron at 3, 7 and 11 DIV (FIG. 4E) were measured.

5. Functional Analysis

a. Calcium Staining

[0048] Calcium imaging was used to confirm synaptic connectivity between neurons. After staining with the calcium dye X-rhod-1, neurons were selected and their intensity tracked under a confocal microscope with a sampling rate of 1.56 s/frame. The chemical stimulation using potassium chloride, resulted in a significant increase of calcium events in neurons confirming their functionality (FIG. 5).

b. Permeability Tests

[0049] 10 kDa Oregon green 488-dextran (Life technologies) and 70 kDa Texas red Dextran (Sigma) were used as fluorescent tracers to assess the permeability of the endothelial barrier. After 4 or 7 days of endothelial cell culture in the device, the cell culture media was removed from the I/O reservoirs and dextran in EGM-2 was injected through the fluidic channel containing endothelial cells (lumen side) simultaneously with blank media on the other fluidic channel to maintain the equilibrium of hydrostatic pressure in the device.

Fluorescence images were captured before the fluorescent solution injection (background) and every min after the injection for 20 min with an 1X81 inverted microscope (Olympus, Tokyo, Japan) using a 4x objective. At least three devices for each condition were used for the imaging and data analysis. Permeability coefficients P of 10 kDa and 70 kDa dextrans were computed for the monoculture of endothelial cells. FIG. 3G compares the mean P for hCMEC/D3 and HUVEC at 4 DIV. The mean P ± SEM for hCMEC/D3 for 70 kDa and 10 kDa dextrans were 1.29 ± 0.15 x 10 "5 cm/s and 2.16 ± 0.28 χ 10 "5 cm/s (n=15, where n is the number of regions chosen for measurements), respectively. The mean P ± SEM for HUVEC for 70 kDa and 10 kDa dextrans were 4.23 ± 0.62 χ 10 "5 cm/s and 6.58 ± 0.55x 10 "5 cm/s (n=19), respectively. These permeability coefficients of 10 kDa and 70 kDa dextrans showed that the barriers formed by HUVEC and hCMEC/D3 were selective according to size where barriers presented statistically significant higher permeability to tracers of lower molecular weight.

[0050] However, the HUVEC monolayer exhibited a higher permeability coefficient compared to hCMEC/D3 and was not stable in the device for more than 5-6 days, while the hCMEC/D3 monolayer remained intact and stable for up to 7-8 days after seeding. Thus, a permeability test was also performed on hCMEC/D3 at 7 DIV and the P was compared with the tests at 4 DIV (Fig. 3H). The mean P ± SEM for hCMEC/D3 at 7 DIV for 70 kDa and 10 kDa dextrans were 0.33 ± 0.03 χ 10 "5 cm/s (n=31) and 1.23 ± 0.12 x 10 "5 cm/s (n=27), respectively. Thus, the initial seeding density and the culture period in-vitro are important factors for the permeability of endothelial cells.

[0051] Moreover, fluorescent dextran assays to measure the endothelial barrier were performed in the case of co-culture of endothelial cells with neurons and astrocytes. Figure 31 compares the mean P for hCMEC/D3 and HUVEC in co-culture condition. The mean P ±

SEM for hCMEC/D3 for 70 kDa and 10 kDa dextrans were 1.00 ± 0.13 χ 10 "5 cm/s and 2.1 ± 0.24 10 "5 cm/s (n=17), respectively. The mean P ± SEM for HUVEC for 70 kDa and 10 kDa dextrans were 2.66 ± 0.20 χ 10 "5 cm/s and 3.49 ± 0.32 χ 10 "5 cm/s (n=12), respectively.

[0052] References

[0053] 1. Abbott, N.J., Patabendige, A.A.K., Dolman, D.E.M., Yusof, S.R., and

Begley, D.J. (2010). Structure and function of the blood-brain barrier. Neurobiol Dis 37, 13- 25. [0054] 2. Chan, J.M., Zervantonakis, I.K., Rimchala, T., Polacheck, W.J.,

Whisler, J., and Kamm, R.D. (2012). Engineering of in-vitro 3D capillary beds by self- directed angiogenic sprouting. PLoS ONE 7, e50582.

[0055] 3. Farahat, W.A., Wood, L.B., Zervantonakis, I.K., Schor, A., Ong, S.,

Neal, D., Kamm, R.D., and Asada, H.H. (2012). Ensemble analysis of angiogenic growth in three-dimensional microfluidic cell cultures. PLoS ONE 7, e37333.

[0056] 4. Zervantonakis, I.K., Hughes-Alford, S.K., Charest, J.L., Condeelis,

J.S., Gertler, F.B., and Kamm, R.D. (2012). Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. Proc Natl Acad Sci USA 109, 13515-

13520.

[0057] 5. Aref, A.R., Huang, R.Y.-J., Yu, W., Chua, K.-N., Sun, W., Tu, T.-Y.,

Bai, J., Sim, W.-J., Zervantonakis, I.K., Thiery, J.P., et al. (2013). Screening therapeutic EMT blocking agents in a three-dimensional microenvironment. Integr Biol (Camb) 5, 381-389.

[0058] 6. K. Vu, B. Weksler, I. Romero, P. O. Couraud, and A. Gelli,

"Immortalized Human Brain Endothelial Cell Line HCMEC/D3 as a Model of the Blood- Brain Barrier Facilitates In-Vitro Studies of Central Nervous System Infection by

Cryptococcus neoformans," Eukaryotic Cell, vol. 8, no. 11, pp. 1803-1807, Oct. 2009.

[0059] 7. B. Poller, H. Gutmann, S. Krahenbuhl, B. Weksler, I. Romero, P.-O.

Couraud, G. Tuffin, J. Drewe, and J. Huwyler, "The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies," Journal of Neurochemistry, vol. 107, no. 5, pp. 1358-1368, Dec. 2008.

[0060] 8. C. J. Czupalla, S. Liebner, and K. Devraj, "In-vitro models of the blood-brain barrier.," Methods Mol. Biol, vol. 1135, pp. 415-437, 2014.

[0061] 9. G. Li, M. J. Simon, L. M. Cancel, Z.-D. Shi, X. Ji, J. M. Tarbell, B.

Morrison, and B. M. Fu, "Permeability of Endothelial and Astrocyte Cocultures: In-Vitro Blood-Brain Barrier Models for Drug Delivery Studies," Ann Biomed Eng, vol. 38, no. 8, pp. 2499-2511, Apr. 2010.

[0062] 10. B. Prabhakarpandian, M.-C. Shen, J. B. Nichols, I. R. Mills, M.

Sidoryk-Wegrzynowicz, M. Aschner, and K. Pant, "SyM-BBB: a microfluidic blood brain barrier model," Lab Chip, vol. 13, no. 6, p. 1093, 2013.

[0063] 11. L. Cucullo, P.-O. Couraud, B. Weksler, I.-A. Romero, M. Hossain, E.

Rapp, and D. Janigro, "Immortalized human brain endothelial cells and flow-based vascular modeling: a marriage of convenience for rational neurovascular studies.," J. Cereb. Blood Flow Metab., vol. 28, no. 2, pp. 312-328, Feb. 2008.

[0064] 12. C. M. Zehendner, R. White, J. Hedrich, and H. J. Luhmann, "A neurovascular blood-brain barrier in-vitro model.," Methods Mol. Biol., vol. 1135, pp. 403- 413, 2014.

[0065] 13. Q. Xue, Y. Liu, H. Qi, Q. Ma, L. Xu, W. Chen, G. Chen, and X. Xu,

"A Novel Brain Neurovascular Unit Model with Neurons, Astrocytes and Microvascular Endothelial Cells of Rat," Int. J. Biol. Sci., vol. 9, no. 2, pp. 174-189.

[0066] 14. L. M. Griep, F. Wolbers, B. de Wagenaar, P. M. ter Braak, B. B.

Weksler, I. A. Romero, P. O. Couraud, I. Vermes, A. D. van der Meer, and A. van den Berg, "BBB ON CHIP: microfluidic platform to mechanically and biochemically modulate blood- brain barrier function," Biomed Microdevices, vol. 15, no. 1, pp. 145-150, Sep. 2012.

[0067] 15. J. H. Yeon, D. Na, K. Choi, S.-W. Ryu, C. Choi, and J.-K. Park,

"Reliable permeability assay system in a microfluidic device mimicking cerebral

vasculatures.," Biomed Microdevices, vol. 14, no. 6, pp. 1141-1148, Dec. 2012.

[0068] 16. R. Booth and H. Kim, "Characterization of a microfluidic in-vitro model of the blood-brain barrier (μΒΒΒ)," Lab Chip, vol. 12, no. 10, p. 1784, 2012.

[0069] 17. E. Urich, C. Patsch, S. Aigner, M. Graf, R. Iacone, and P.-O.

Freskgard, "Multicellular Self-Assembled Spheroidal Model of the Blood Brain Barrier," Sci. Rep., vol. 3, Mar. 2013.

[0070] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0071] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.