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WO2017035119A1 - Modèle de barrière hémato-encéphalique dans un système microfluidique 3d de co-culture - Google Patents

Modèle de barrière hémato-encéphalique dans un système microfluidique 3d de co-culture Download PDF

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WO2017035119A1
WO2017035119A1 PCT/US2016/048138 US2016048138W WO2017035119A1 WO 2017035119 A1 WO2017035119 A1 WO 2017035119A1 US 2016048138 W US2016048138 W US 2016048138W WO 2017035119 A1 WO2017035119 A1 WO 2017035119A1
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gel
neuron
posts
astrocyte
row
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Roger Dale Kamm
Dong Liang MA
Eyleen Lay Keow GOH
Giulia ADRIANI
Andrea Pavesi
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National University Of Singapore
Massachusetts Institute Of Technology
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Publication of WO2017035119A1 publication Critical patent/WO2017035119A1/fr

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/081—Coculture with; Conditioned medium produced by cells of the nervous system neurons

Definitions

  • the Blood Brain Barrier 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].
  • 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].
  • 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].
  • fluid flow in the microfluidic platforms was considered to investigate the effect of shear stress on endothelial junction formation [16].
  • 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.
  • BBB Blood Brain Barrier
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. IB is a time line of an experiment, in accordance with a version of the invention.
  • 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 ⁇ .
  • 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
  • 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 ⁇
  • the scale bar in FIG. 2C is 50 ⁇ .
  • 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 ⁇ .
  • 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.
  • 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 ⁇ .
  • 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 ⁇ .
  • 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 ⁇ ; 700-1400 ⁇ ; 1400-2100 ⁇ ).
  • 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.
  • 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.
  • 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.
  • BBB Blood Brain Barrier
  • a microfluidic device consists of two central gel regions flanked by two media channels, as described further relative to FIG.1A, 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.
  • 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.
  • 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).
  • PDMS poly(dimethylsiloxane)
  • 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.
  • 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.
  • each post rows of posts 114, 115 and 116 forms a triangular shape, a trapezoidal shape or a combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • only a single gel region 101 is used, containing a mixture of both astrocytes and neurons.
  • 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.
  • 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.
  • 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.
  • FIGS. IB through 5C are described in further detail in the Experimental section, below, with reference to the Brief Description of the drawings, above.
  • 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.
  • BBB blood brain barrier
  • Advantages of a BBB microfluidic model in accordance with a version of the invention can include:
  • 3D microenvironment optimized for multi-cellular co-culture e.g., endothelial cells, neurons and astrocytes growth;
  • 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.
  • 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.
  • human induced pluripotent stem cells ⁇ e.g., provided by Cellular Dynamics International, Inc. of Madison, WI, U.S.A.
  • patient-derived cells for a personalized BBB model.
  • 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.
  • PDMS poly(dimethylsiloxane)
  • Sylgard 184 Silicone elastomer kit Dow Corning, Midland, MI, USA
  • 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 ⁇ 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).
  • 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).
  • PDL Poly-D-lysine
  • 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 ⁇ 10 6 cells/ml or 5 ⁇ 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.
  • the fluidic channels were incubated with collagen (100 ⁇ g/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).
  • Doublecortin is a microtubule-associated protein specific for immature neurons, and antibody against DCX allows a clear visualization of the cell body and neurites.
  • GFAP glial fibrillary acidic protein
  • FIG. 2A 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.
  • 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.
  • 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 ⁇ ; 700-1400 ⁇ ; 1400-2100 ⁇ were calculated at 3, 7 and 11 DIV (FIG. 4).
  • 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.
  • 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 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.
  • 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 initial seeding density and the culture period in-vitro are important factors for the permeability of endothelial cells.
  • HCMEC/D3 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

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Abstract

La présente invention concerne un modèle de barrière hémato-encéphalique (BBB) efficace in vitro consistant en des cellules clés de la BBB - à savoir, des neurones, des astrocytes et des cellules endothéliales - qui permet de récapituler la BBB in vivo et d'apporter des éclaircissements sur des contributionsde chaque type de cellule individuelle. Un système microfluidique pour la modélisation de la barrière hémato-encéphalique comprend un substrat optiquement transparent, ledit substrat comprenant : (i) au moins un canal de fluide ; (ii) un premier canal de gel comprenant une première région de gel ; (iii) un second canal de gel comprenant une seconde région de gel ; et (iv) au moins une rangée de montants.
PCT/US2016/048138 2015-08-24 2016-08-23 Modèle de barrière hémato-encéphalique dans un système microfluidique 3d de co-culture WO2017035119A1 (fr)

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EP3365424A4 (fr) * 2015-10-19 2019-04-17 Emulate, Inc. Modèle microfluidique de la barrière hémato-encéphalique
US12042791B2 (en) 2016-01-12 2024-07-23 Cedars-Sinai Medical Center Method of osteogenic differentiation in microfluidic tissue culture systems
US11473061B2 (en) 2016-02-01 2022-10-18 Cedars-Sinai Medical Center Systems and methods for growth of intestinal cells in microfluidic devices
US11913022B2 (en) 2017-01-25 2024-02-27 Cedars-Sinai Medical Center In vitro induction of mammary-like differentiation from human pluripotent stem cells
US10457717B2 (en) 2017-02-17 2019-10-29 Denali Therapeutics Inc. Engineered polypeptides
US11795232B2 (en) 2017-02-17 2023-10-24 Denali Therapeutics Inc. Engineered transferrin receptor binding polypeptides
US12162948B2 (en) 2017-02-17 2024-12-10 Denali Therapeutics Inc. Methods of engineering transferrin receptor binding polypeptides
US11612150B2 (en) 2017-02-17 2023-03-28 Denali Therapeutics Inc. Transferrin receptor transgenic models
US10143187B2 (en) 2017-02-17 2018-12-04 Denali Therapeutics Inc. Transferrin receptor transgenic models
US11732023B2 (en) 2017-02-17 2023-08-22 Denali Therapeutics Inc. Engineered polypeptides
US11912778B2 (en) 2017-02-17 2024-02-27 Denali Therapeutics Inc. Methods of engineering transferrin receptor binding polypeptides
US11767513B2 (en) 2017-03-14 2023-09-26 Cedars-Sinai Medical Center Neuromuscular junction
US11414648B2 (en) 2017-03-24 2022-08-16 Cedars-Sinai Medical Center Methods and compositions for production of fallopian tube epithelium
US12161676B2 (en) 2018-03-23 2024-12-10 Cedars-Sinai Medical Center Methods of use of islet cells
US12241085B2 (en) 2018-04-06 2025-03-04 Cedars-Sinai Medical Center Human pluripotent stem cell derived neurodegenerative disease models on a microfluidic chip
US11981918B2 (en) 2018-04-06 2024-05-14 Cedars-Sinai Medical Center Differentiation technique to generate dopaminergic neurons from induced pluripotent stem cells
CN108823145B (zh) * 2018-06-01 2022-04-05 武汉轻工大学 一种人脑微血管生成模拟血脑屏障的体外构建方法
CN108823145A (zh) * 2018-06-01 2018-11-16 武汉轻工大学 一种人脑微血管生成模拟血脑屏障的体外构建方法
EP3839038A4 (fr) * 2018-09-19 2022-06-15 Cellartgen Inc. Dispositif microfluidique de simulation cérébrovasculaire et système de simulation de barrière hémato-encéphalique à haut rendement le comprenant
JPWO2020090771A1 (ja) * 2018-10-29 2021-09-24 国立大学法人 東京大学 人工多層組織培養デバイスと人工多層組織の製造方法
JP7388724B2 (ja) 2018-10-29 2023-11-29 国立大学法人 東京大学 人工多層組織培養デバイスと人工多層組織の製造方法
WO2020090771A1 (fr) * 2018-10-29 2020-05-07 国立大学法人東京大学 Dispositif de culture de tissu multicouche artificiel, et procédé de production de tissu multicouche artificiel
US11198842B1 (en) 2019-02-22 2021-12-14 University Of South Florida Microfluidic-coupled in vitro model of the blood-brain barrier
EP3943951A4 (fr) * 2019-03-20 2023-06-28 University Public Corporation Osaka Dispositif fluidique
US20220169970A1 (en) * 2019-03-20 2022-06-02 University Public Corporation Osaka Fluidic device
EP4057001A4 (fr) * 2019-12-05 2023-12-13 Cha University Industry-Academic Cooperation Foundation Puce nerveuse biomimétique pour évaluer l'efficacité et la toxicité sur un nerf, et son utilisation
WO2021216848A1 (fr) * 2020-04-22 2021-10-28 The Board Of Trustees Of The Leland Stanford Junior University Puces microfluidiques et systèmes microphysiologiques les utilisant
WO2021224329A1 (fr) 2020-05-08 2021-11-11 Technische Universität Wien Dispositif microfluidique
EP3907275A1 (fr) * 2020-05-08 2021-11-10 Technische Universität Wien Dispositif microfluidique
CN114181833B (zh) * 2021-12-21 2023-04-25 上海交通大学医学院附属瑞金医院 一种基于纤维蛋白凝胶模拟病理性血脑屏障的仿生微流控芯片及其构建方法
CN114181833A (zh) * 2021-12-21 2022-03-15 上海交通大学医学院附属瑞金医院 一种基于纤维蛋白凝胶模拟病理性血脑屏障的仿生微流控芯片及其构建方法

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