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WO2020081288A1 - Dispositifs microfluidiques et procédés incorporant des constructions de tissue tridimensionnelles organisées - Google Patents

Dispositifs microfluidiques et procédés incorporant des constructions de tissue tridimensionnelles organisées Download PDF

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Publication number
WO2020081288A1
WO2020081288A1 PCT/US2019/055174 US2019055174W WO2020081288A1 WO 2020081288 A1 WO2020081288 A1 WO 2020081288A1 US 2019055174 W US2019055174 W US 2019055174W WO 2020081288 A1 WO2020081288 A1 WO 2020081288A1
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cell suspension
suspension region
microfluidic device
fluid
fluid channel
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Mehdi Nikkhah
Jaimeson VELDHUIZEN
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Arizona State University ASU
Arizona State University Downtown Phoenix campus
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Arizona State University ASU
Arizona State University Downtown Phoenix campus
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Priority to US16/650,346 priority Critical patent/US20210054321A1/en
Publication of WO2020081288A1 publication Critical patent/WO2020081288A1/fr
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    • C—CHEMISTRY; METALLURGY
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid

Definitions

  • This disclosure relates to microfluidic devices, including devices incorporating highly organized (e.g., aligned) three-dimensional, live tissue constructs cells, and associated methods of fabricating and using such devices.
  • Cardiac ischemia including acute myocardial infarction (MI,“heart attack”) and chronic ischemic heart disease (chronic IHD), are highly progressive biological disorders that can ultimately lead to catastrophic heart failure and death.
  • MI acute myocardial infarction
  • chronic IHD chronic ischemic heart disease
  • Hypoxia has been implicated as the major regulator of ischemia-induced cardiac injury, through triggering of a multitude of molecular/cellular signaling cascades.
  • acute hypoxia leads to necrosis of cardiomyocytes (CMs) that induces upregulation of inflammatory cytokines and cellular paracrine signaling, followed by contractile dysfunction and adverse remodeling of the myocardium.
  • CMs cardiomyocytes
  • CMs and neighboring interstitial cells adds a higher level of complexity to the mechanism of disease progression.
  • the physiological and pathophysiological responses of the heart in healthy and diseased states have been the subject of intensive studies using gold standard animal models.
  • animal models may lack physiological relevancy to humans, with the inability to precisely control micro environmental cues for cellular and molecular level studies. This has led to a major knowledge gap in full understanding of the molecular/cellular basis of cardiac injury.
  • the inability of animal models to fully recapitulate human physiology often leads to failures in bench to bedside therapeutics development, highlighting the critical need to develop alternative platform technologies using human cardiac cells that would have translational advantages over animal models.
  • CMs or stem-cell-derived cardiac cells are encapsulated in natural or synthetic hydrogels, representative of the native extracellular matrix (ECM) of myocardium.
  • ECM extracellular matrix
  • elastomers are used to create intricate microchannels and entities that house these hydrogel-encapsulated tissues.
  • the few devices that do have topographical cues to induce cellular alignment fail to provide a biomimetic 3D human tissue architecture and precise mechanistic insight into the phenotypic signatures of cardiac cells and the pathophysiological alterations of myocardium from healthy to diseased state.
  • a microfluidic device that incorporates a highly organized co culture of live cells within a microengineered platform, by which the architecture and cellular constituents of an organ or other native tissue environment may be modeled over a long period of culture for biological and/or pharmacological studies.
  • Vertical posts e.g., microposts
  • Such a microfluidic device can complement animal studies in recapitulating pathophysiological characteristics of disease.
  • such a microfluidic device When populated with cardiac cells, such a microfluidic device provides a three-dimensional (3D) biomimetic human cardiac tissue model that enables the study of pathophysiological events involved in the transition of healthy to diseased cardiac tissue, to better inform therapeutic strategies and functional outcomes in cardiac -based therapies.
  • 3D three-dimensional
  • Other types of cells may be used in certain embodiments. Methods of fabricating and using such microfluidic devices are also provided.
  • the disclosure relates to a microfluidic device comprising: a cell suspension region; a plurality of linear arrays of vertical posts arranged within the cell suspension region; at least one hydrogel insertion port arranged upstream of the cell suspension region; at least one sample extraction port arranged downstream of the cell suspension region; a first fluid channel arranged proximate to the cell suspension region; a first fluid-permeable boundary wall arranged between the first fluid channel and the cell suspension region, and forming a first lateral boundary of the cell suspension region; a second fluid channel arranged proximate to the cell suspension region; a second fluid-permeable boundary wall arranged between the second fluid channel and the cell suspension region, and forming a second lateral boundary of the cell suspension region; a first fluid channel inlet port and a first fluid channel outlet port in fluid communication with the first fluid channel; and a second fluid channel inlet port and a second fluid channel outlet port in fluid communication with the second fluid channel; wherein the cell suspension region is enclosed from above and below.
  • the cell suspension region contains a 3D hydrogel matrix having cells embedded therein and arranged in contact with the plurality of linear arrays of vertical posts.
  • the cells comprise at least one of: animal- or human- derived cardiac cells, animal- or human-derived neural cells, or animal- or human-derived skeletal muscle cells.
  • each vertical post of the plurality of linear arrays of vertical posts includes a tapered leading edge and a tapered trailing edge. In certain embodiments, each vertical post of the plurality of linear arrays of vertical posts includes a length, from the tapered leading edge to the tapered trailing edge, in a range of from 200 microns to 500 microns.
  • vertical posts of the plurality of linear arrays of vertical posts are spaced apart from adjacent vertical posts by a lengthwise dimension in a range of from 100 to 300 microns, and by a widthwise dimension in a range of from 100 to 300 microns.
  • the first fluid-permeable boundary wall comprises a first array of trapezoidal posts
  • the second fluid-permeable boundary wall comprises a second array of trapezoidal posts.
  • the first array of trapezoidal posts comprises a first plurality of linearly arranged trapezoidal posts, with each trapezoidal post of the first plurality of linearly arranged trapezoidal posts comprising a short end arranged closer to the cell suspension region than to the first fluid channel; and the second array of trapezoidal posts comprises a second plurality of linearly arranged trapezoidal posts, with each trapezoidal post of the second plurality of linearly arranged trapezoidal posts comprising a short end arranged closer to the cell suspension region than to the second fluid channel.
  • the at least one sample extraction port comprises first and second sample extraction ports that are laterally offset relative to one another.
  • the microfluidic device further comprises a first sample extraction channel arranged between the cell suspension region and the first sample extraction port; and a second sample extraction channel arranged between the cell suspension region and the second sample extraction port; wherein the first sample extraction channel is laterally offset relative to the second sample extraction channel.
  • the at least one hydrogel insertion port comprises a first hydrogel insertion port and a second hydrogel insertion port that are laterally offset relative to one another; and the microfluidic device further comprises a first hydrogel insertion channel arranged between the first hydrogel insertion port and the cell suspension region, and a second hydrogel insertion channel arranged between the second hydrogel insertion port and the cell suspension region, wherein the first hydrogel insertion channel is laterally offset relative to the second hydrogel insertion channel.
  • the microfluidic device further comprises endothelial cells seeded in at least one of the first fluid channel or the second fluid channel.
  • one or more of the first fluid channel, the second fluid channel, or the cell suspension region comprises a height dimension and/or width dimension of less than 500 microns.
  • the microfluidic device further comprises at least one electrode arranged in conductive electrical communication with the cell suspension region.
  • the disclosure relates to a microfluidic device comprising: a cell suspension region; at least one hydrogel insertion port arranged upstream of the cell suspension region; a plurality of sample extraction ports arranged downstream of and in fluid communication with the cell suspension region, wherein each sample extraction port of the plurality of sample extraction ports is laterally offset relative to at least one other sample extraction port of the plurality of sample extraction ports; a first fluid channel arranged proximate to the cell suspension region; a first fluid-permeable boundary wall arranged between the first fluid channel and the cell suspension region, and forming a first lateral boundary of the cell suspension region; a second fluid channel arranged proximate to the cell suspension region; a second fluid-permeable boundary wall arranged between the second fluid channel and the cell suspension region, and forming a second lateral boundary of the cell suspension region; a first fluid channel inlet port and a first fluid channel outlet port in fluid communication with the first fluid channel; and a second fluid channel inlet port and a second fluid channel
  • each vertical post of the plurality of linear arrays of vertical posts includes a tapered leading edge and a tapered trailing edge. In certain embodiments, each vertical post of the plurality of linear arrays of vertical posts includes a length, from the tapered leading edge to the tapered trailing edge, in a range of from 200 microns to 500 microns.
  • vertical posts of the plurality of linear arrays of vertical posts are spaced apart from adjacent vertical posts by a lengthwise dimension in a range of from 100 to 300 microns, and by a widthwise dimension in a range of from 100 to 300 microns.
  • the cell suspension region contains a 3D hydrogel matrix having cells embedded therein and arranged in contact with the plurality of linear arrays of vertical posts.
  • the first fluid-permeable boundary wall comprises a first array of trapezoidal posts
  • the second fluid-permeable boundary wall comprises a second array of trapezoidal posts.
  • the first array of trapezoidal posts comprises a first plurality of linearly arranged trapezoidal posts, with each trapezoidal post of the first plurality of linearly arranged trapezoidal posts comprising a short end arranged closer to the cell suspension region than to the first fluid channel; and the second array of trapezoidal posts comprises a second plurality of linearly arranged trapezoidal posts, with each trapezoidal post of the second plurality of linearly arranged trapezoidal posts comprising a short end arranged closer to the cell suspension region than to the second fluid channel.
  • the plurality of sample extraction ports comprises a first sample extraction port and a second sample extraction port; and the microfluidic device further comprises a first sample extraction channel arranged between the cell suspension region and the first sample extraction port, and a second sample extraction channel arranged between the cell suspension region and the second sample extraction port, wherein the first sample extraction channel is laterally offset relative to the second sample extraction channel.
  • the at least one hydrogel insertion port comprises a first hydrogel insertion port and a second hydrogel insertion port that are laterally offset relative to one another; and the microfluidic device further comprises a first hydrogel insertion channel arranged between the first hydrogel insertion port and the cell suspension region, and a second hydrogel insertion channel arranged between the second hydrogel insertion port and the cell suspension region, wherein the first hydrogel insertion channel is laterally offset relative to the second hydrogel insertion channel.
  • the microfluidic device further comprises endothelial cells seeded in at least one of the first fluid channel or the second fluid channel.
  • one or more of the first fluid channel, the second fluid channel, and the cell suspension region comprises a height dimension and/or width dimension of less than 500 microns.
  • the microfluidic device further comprises at least one electrode arranged in conductive electrical communication with the cell suspension region.
  • the disclosure relates to a method for fabricating a microfluidic device, the method comprising: supplying a suspension of cells in a hydrogel solution through at least one hydrogel insertion port into a cell suspension region of a microfluidic device to contact a plurality of linear arrays of vertical posts arranged within the cell suspension region, wherein the cell suspension region is enclosed from above and below, and is laterally bounded by first and second fluid-permeable boundary walls; and polymerizing the hydrogel solution within the cell suspension region to form a 3D hydrogel matrix having cells embedded therein and in contact with the plurality of linear arrays of vertical posts.
  • the cells embedded in the 3D hydrogel matrix comprise at least one of: animal- or human-derived cardiac cells, animal- or human-derived neural cells, or animal- or human-derived skeletal muscle cells.
  • the polymerizing of the hydrogel solution comprises at least one of thermal, chemical, or photonic polymerization.
  • the disclosure relates to a method for using a microfluidic device that includes a 3D hydrogel matrix having cells embedded therein contained within a cell suspension region that is laterally bounded by first and second fluid-permeable boundary walls, wherein first and second sample extraction ports are laterally offset relative to one another and arranged downstream of the cell suspension region.
  • Such method comprises: supplying a first fluid containing at least one first component susceptible to interaction with the cells into a first fluid channel, wherein the first fluid-permeable boundary wall is arranged between the first fluid channel and the cell suspension region, to cause the at least one first component to contact cells within the 3D hydrogel matrix; supplying a second fluid containing at least one second component susceptible to interaction with the cells into a second fluid channel, wherein the second fluid-permeable boundary wall is arranged between the second fluid channel and the cell suspension region, to cause the at least one second component to contact cells within the 3D hydrogel matrix; and releasing at least some cells from the 3D hydrogel matrix to cause a first group of cells to flow through the first sample extraction port, and to cause a second group of cells to flow through the second sample extraction port.
  • the releasing of at least some cells from the 3D hydrogel matrix comprises enzymatic digestion of at least a portion of the 3D hydrogel matrix.
  • the at least one first component is compositionally different from the at least one second component.
  • the at least one first component comprises a different concentration than the at least one second component.
  • the at least one first component and the at least one second component are independently selected from the group consisting of: cytokines, growth factors, oxygen, drugs, toxins, nanoparticles, and chemical agents.
  • FIG. 1 is a schematic diagram showing a microfluidic device with encapsulated cardiac tissue providing a model for healthy and diseased tissue states.
  • FIG. 2 is a top view of at least a portion of an example microfluidic device.
  • FIG. 3 is a top view of at least a portion of another example microfluidic device.
  • FIG. 4 is a schematic diagram showing steps of a method involving photolithography and replica molding for fabricating a microfluidic device as disclosed herein.
  • FIG. 5 is a perspective view of a microfluidic device according to FIG. 2.
  • FIG. 6 is a top plan view of a microfluidic device according to FIGS. 2 and 5 arranged proximate to a U.S. penny (one cent coin) to show the size of the microfluidic device.
  • FIG. 7 is a table of experimental variables that were tested with a platform to promote their optimization, with such variables including hydrogel composition, micropost shape, micropost length, widthwise post spacing, and lengthwise post spacing.
  • FIGS. 8-11 provide phase contrast images for microfluidic devices with different combinations of cardiomyocytes (CM): cardiac fibroblasts (CF) cell ratios and geometries for vertical posts.
  • CM cardiomyocytes
  • CF cardiac fibroblasts
  • FIG. 12 is a still image obtained from a contraction video for a 4: 1 cell ratio of CM:CF.
  • FIG. 13 is a still image obtained from a contraction video for a 8:1 cell ratio of CM:CF.
  • FIGS. 14A-17B provide cytoskeletal staining images and their color- inverted representations of rat cardiac tissue between vertical posts within microfluidic devices according to four designs.
  • FIG. 18A provides a Z-stacked image of immunostained cell matrices within a microfluidic device at 20X magnification to demonstrate the three-dimensional (3D) character of the tissue within the cell suspension region.
  • FIG. 18B is a color-inverted representation of the Z-stacked image of FIG. 18A.
  • FIG. 19A provides a Z-stacked image of immunostained cell matrices within a microfluidic device at 40X magnification to demonstrate the 3D character of the tissue within the cell suspension region.
  • FIG. 19B is a color-inverted representation of the Z-stacked image of FIG. 19A.
  • FIG. 20 is a bar chart depicting calculated thickness of aligned tissues within microfluidic devices according to four designs for three experiments with triplicate samples.
  • FIG. 21 is a plot of orientation of 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei of cells within microfluidic devices according four designs.
  • DAPI 4',6-diamidino-2-phenylindole
  • FIGS. 22-25 provide images of cardiac marker staining of rat cardiac tissue between vertical posts for four designs of the microfluidic device as disclosed herein.
  • FIG. 26A is a 40X magnification image of a tissue within a microfluidic device according to one design.
  • FIG. 26B is a color inverted copy of the image of FIG. 26A.
  • FIG. 27 is a line graph plotting representative beating signals of a tissue for microfluidic devices according to four designs.
  • FIGS. 28-31B represent items obtained from preliminary studies performed on a co culture of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and human CFs in hydrogel-encapsulated tissue within a cell suspension region of a microfluidic device as disclosed herein over a period of 6 days.
  • hiPSC-CMs human induced pluripotent stem cell derived cardiomyocytes
  • CFs hydrogel-encapsulated tissue within a cell suspension region of a microfluidic device as disclosed herein over a period of 6 days.
  • FIGS. 32A-33C represent items obtained from additional studies performed on a co culture of hiPSC-CMs and human embryonic stem cell-derived cardiomyocytes (hESC-CMs), together referred to as human pluripotent stem cells (hPSCs), in hydrogel-encapsulated tissue within a cell suspension region of a microfluidic device as disclosed herein.
  • hiPSC-CMs human embryonic stem cell-derived cardiomyocytes
  • hPSCs human pluripotent stem cells
  • FIGS. 34A-34C provide images of expression of cardiac markers of hiPSC-derived cardiac tissues inside a microfluidic device and devices without posts.
  • a microfluidic device disclosed herein incorporates an aligned three-dimensional (3D) tissue constructs of live cells. Such a device enables formation of a 3D biomimetic human cardiac tissue model. Other types of cells, such as neural, embryonic stem cells, or skeletal cells, may be used in certain embodiments. Vertical posts (e.g., microposts) may be provided to induce alignment of hydrogel-encapsulated tissues in a cell suspension region of a microfluidic device.
  • FIG. 1 is a schematic diagram showing a microfluidic device 10 (e.g., a microfluidic chip) with encapsulated cardiac tissue 12, providing a model for healthy and diseased tissue states.
  • a first portion 14 of the encapsulated cardiac tissue 12 corresponds to a normal myocardium, with an extracellular matrix (ECM) containing cardiac fibroblasts and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).
  • ECM extracellular matrix
  • hiPSC-CMs human induced pluripotent stem cell derived cardiomyocytes
  • a second portion 16 of the encapsulated cardiac tissue 12 corresponds to unhealthy/scar tissue contained in an ECM.
  • the first and second portions 14, 16 of the encapsulated cardiac tissue 12 may have initially been the same in character, but diverged in health upon exposure to different conditions, growth factors, chemical agents, and/or drugs.
  • FIG. 2 is a top view of at least a portion of an example microfluidic device 10, including a cell suspension region 18 (at center) containing a plurality of (e.g., four) linear arrays 20 of vertical posts 22 each having a modified elliptical shape, with a tapered leading edge 24 and a tapered trailing edge 26.
  • Each vertical post 22 is spaced apart from at least one adjacent vertical post 22 in a widthwise direction by a widthwise post spacing ( wPS ), and is spaced apart from at least one adjacent vertical post 22 in a lengthwise direction by a lengthwise post spacing (IPS).
  • wPS widthwise post spacing
  • IPS lengthwise post spacing
  • Two hydrogel insertion ports (e.g., a first hydrogel insertion port 28 and a second hydrogel insertion port 30, which are laterally offset relative to one another) lead to two laterally offset hydrogel insertion channels (e.g., a first hydrogel insertion channel 32 and a second hydrogel insertion channel 34) arranged upstream of the cell suspension region 18.
  • Two sample extraction channels (e.g., a first sample extraction channel 36 and a second sample extraction channel 38, which are laterally offset relative to one another) lead to two sample extraction ports (e.g., a first sample extraction port 40 and a second sample extraction port 42) and are arranged downstream of the cell suspension region 18.
  • a first fluid channel 44 and a second fluid channel 46 are arranged along opposing sides of the cell suspension region 18.
  • the first fluid channel 44 extends between a first fluid channel inlet port 48 and a first fluid channel outlet port 50
  • the second fluid channel 46 extends between a second fluid channel inlet port 52 and a second fluid channel outlet port 54.
  • a first fluid-permeable boundary wall 56 is arranged between the first fluid channel 44 and the cell suspension region 18, and forms a first lateral boundary of the cell suspension region 18.
  • a second fluid-permeable boundary wall 58 is arranged between the second fluid channel 46 and the cell suspension region 18, and forms a second lateral boundary of the cell suspension region 18.
  • Each of the first and the second fluid-permeable boundary wall 56, 58 includes a linear array of trapezoidal posts 60, with each trapezoidal post 60 comprising a short end 62 arranged closer to the cell suspension region 18 than to the adjacent first fluid channel 44 or second fluid channel 46.
  • each trapezoidal post 60 may be spaced apart from at least one adjacent trapezoidal post 60 by a distance of 20-30 microns (pm).
  • the trapezoidal posts 60 ensure protection of the cell-embedded hydrogel matrix from fluidic shear stress applied by flow of fluids through the first fluid channel 44 and the second fluid channel 46, while allowing the diffusion of nutrients through the hydrogel matrix.
  • the edges of the hydrogel insertion channels 32, 34 are tapered at a mouth of the cell suspension region 18.
  • FIG. 3 is a top view of at least a portion of another example microfluidic device 10, including a cell suspension region 18 (at center) containing a plurality of (e.g., four) linear arrays 20 of vertical posts 22 each having a stretched hexagonal shape, with a tapered leading edge 24 and a tapered trailing edge 26.
  • the microfluidic device 10 of FIG. 3 is substantially the same as the microfluidic device 10 of FIG. 2.
  • each vertical post 22 may include a length, from the tapered leading edge 24 to the tapered trailing edge 26, in a range of from 200 microns to 500 pm. Moreover, in certain embodiments, each vertical post 22 may be spaced apart from at least one other adjacent vertical post 22 by a lengthwise dimension (e.g., lengthwise post spacing (IPS)) in a range of from 100 to 300 pm (or in a subrange of from 100 to 250 pm, or from 150 to 300 pm, or from 150 to 250 pm, or from 200 to 300 pm). In certain embodiments, each vertical post 22 may have a length: width ratio in a range of from about 2: 1 to about 10:1, or in a subrange of from about 3:1 to about 8:1, or in a subrange of from about 4:1 to about 7:1.
  • IPS lengthwise post spacing
  • first hydrogel insertion port 28 and the second hydrogel insertion port 30 permits hydrogel solution and cells material to be supplied to the cell suspension region 18 and distributed across a width of the cell suspension region 18.
  • a suspension of cells in a hydrogel solution may be supplied through the hydrogel insertion ports 28, 30 to contact the linear arrays 20 of vertical posts 22 within the cell suspension region 18.
  • the hydrogel solution may be polymerized within the cell suspension region 18 to form a 3D hydrogel matrix having cells embedded therein and in contact with the plurality of linear arrays 20 of vertical posts 22.
  • Such polymerization may be accomplished by at least one of thermal, chemical, or photonic polymerization.
  • the cells embedded in the hydrogel matrix may include at least one of: animal- or human-derived cardiac cells, animal- or human-derived neural cells, or animal- or human-derived skeletal muscle cells.
  • Collagen type I hydrogel may be used for encapsulation of tissue (e.g., the cardiac tissue 12 of FIG. 1) and construction of the 3D microenvironment, with hydrogel concentration in the range as listed in FIG. 7.
  • tissue e.g., the cardiac tissue 12 of FIG. 1
  • Other types of hydrogels known in the art such as (but not limited to) gelatin and hyaluronic acid, could be used for establishing 3D microenvironments.
  • CMs neonatal rat cardiomyocytes
  • CFs cardiac fibroblasts
  • the hydrogel-encapsulated cells can be injected into the cell suspension region 18 through the hydrogel insertion ports 28, 30 (definable by coring through polydimethylsiloxane (PDMS) of the microfluidic device 10) and associated hydrogel insertion channels 32, 34.
  • PDMS polydimethylsiloxane
  • a microfluidic device 10 may be placed in an incubator for 30 minutes at 37°C in a 5% C0 2 atmosphere to allow for gel polymerization. Thereafter, the first fluid channel 44 and the second fluid channel 46 may be filled with media for passage through the fluid-permeable boundary walls 56, 58 to permit nutrients to diffuse through the tissue.
  • first fluid channel 44 and the second fluid channel 46 proximate to lateral boundaries of the cell suspension region 18 permits different fluids to be supplied to the first fluid channel 44 and the second fluid channel 46, respectively, wherein such fluids may diffuse through the fluid-permeable boundary walls 56, 58 to interact with different portions of the cells suspended in the cell suspension region 18.
  • “Different fluids” in this context refers to fluids that may differ in composition and/or concentration.
  • fluids may be supplied at different flow rates and adjusted shear stresses to the first fluid channel 44 and the second fluid channel 46.
  • a gradient condition of one or more constituents of such fluids may result in the cell suspension region 18.
  • cells located at different locations e.g., closer to the first fluid channel 44 than the to the second fluid channel 46
  • This may be used to assess whether and how differing presence and/or concentration of one or more constituents of fluids supplied to the first fluid channel 44 and the second fluid channel 46 may affect condition of cells within the cell suspension region 18.
  • sample extraction channels 36, 38 and sample extraction ports 40, 42 downstream of the cell suspension region 18 also permits cells subjected to different conditions to be separately extracted from the microfluidic device 10 to permit such cells to be separately analyzed.
  • 3 may include: supplying a first fluid containing at least one first component susceptible to interaction with the cells into the first fluid channel 44, to cause the first fluid to permeate the first fluid-permeable boundary wall 56 and cause at least one first component to contact cells within the 3D hydrogel matrix (e.g., in a portion of the cell suspension region 18 closer to the first fluid channel 44); and supplying a second fluid containing at least one second component susceptible to interaction with the cells into the second fluid channel 46, to cause the second fluid to permeate the second fluid-permeable boundary wall 58 and cause the at least one second component to contact cells within the 3D hydrogel matrix (e.g., in a portion of the cell suspension region 18 closer to the second fluid channel 46).
  • the first fluid and the second fluid may be independently selected from the group consisting of: saline, cell culture media, air, plasma, serum, whole blood, buffers, and cytokines.
  • the at least one first component and the at least one second component may be independently selected from the group consisting of: cytokines, growth factors, oxygen, drugs, toxins, nanoparticles, and chemical agents.
  • microposts e.g., the vertical posts 22
  • these vertical posts 22 may be in the range of 500-800 pm long, 100-200 pm wide, and spaced with lengthwise post spacing (IPS) of 150-350 pm and widthwise post spacing (vvPS) of 150-300 pm.
  • IPS lengthwise post spacing
  • vvPS widthwise post spacing
  • FIG. 4 is a schematic diagram showing steps of a method 400 involving photolithography and replica molding for fabricating a microfluidic device 10 as disclosed herein.
  • Microfluidic devices 10 as disclosed herein may be fabricated with PDMS using SU-8 photolithography and replica molding technique.
  • the method starts at operation 402, with spin coating SU-8 photoresist 64 on a silicon wafer 66 to a thickness of 200 pm.
  • the coated wafer 66 is exposed to ultraviolet (UV) light through a transparent mask 68 with the microfluidic device 10 design, created using AutoCAD (minimum features of 20 um).
  • UV ultraviolet
  • the SU-8 photoresist 64 is developed, resulting in a negative replica 70 of the microfluidic device 10. This negative replica 70 may be used to produce a positive replica 72 from the PDMS.
  • the wafer 66 is prepared for PDMS replica molding via silanization (e.g., using methyltryichlorosilane (MTCS)) of the surface to reduce attraction between the cast PDMS and the SU-8 features.
  • the PDMS to be cast is prepared through mixing of the base to curing agent at a ratio of 10:1.
  • this solution is poured over the silanized silicon wafer 66, then degassed in a vacuum and baked in an oven for 2 hours at 80°C.
  • the PDMS is peeled from the wafer 66 and, in examples where an array of microfluidic devices 10 are molded together, each microfluidic device 10 in the array is cut.
  • the inlet and outlet ports are cored down to the channels using standard 1-2 mm punches.
  • the first fluid channel inlet port 48 and first fluid channel outlet port 50 are cored down to the first fluid channel 44
  • the second fluid channel inlet port 52 and second fluid channel outlet port 54 are cored down to the second fluid channel 46.
  • a similar process is applied for the hydrogel insertion ports 28, 30 and/or the sample extraction ports 40, 42.
  • the PDMS surfaces of the positive replica 72 are rendered hydrophilic through the use of air plasma, then bonded to glass slides 74 to create microfluidic channels (e.g., the fluid channels 44, 46, the cell suspension region 18, the hydrogel insertion channels 32, 34, and the sample extraction channels 36, 38).
  • the PDMS microfluidic channel platform is then sterilized at 120°C for 20 minutes in a wet cycle, followed by a dry cycle at 120°C for 35 minutes.
  • one or more electrodes may be provided on at least one surface of a substrate, and such electrode(s) may be in conductive electrical communication with an interior of the microfluidic device 10 (e.g., with the cell suspension region 18).
  • one or more electrodes may be used to stimulate tissue (e.g., stimulate beating of cardiac cells).
  • one or more electrodes may be used for reading or sensing signals generated by the tissue.
  • FIG. 5 is a perspective view of a microfluidic device 10 according to FIG. 2, showing the cell suspension region 18 containing a 3D hydrogel matrix 76 (i.e., a Fibrin: Collagen I hydrogel) forming an ECM that contains CFs and CMs, alongside the bordering first fluid channel 44 and second fluid channel 46.
  • FIG. 6 is a top plan view of a microfluidic device 10 according to FIGS. 2 and 5 arranged proximate to a U.S. penny 78 (one cent coin) to show the size of the microfluidic device 10.
  • a 3D hydrogel matrix 76 i.e., a Fibrin: Collagen I hydrogel
  • Microfluidic devices 10 as disclosed herein have been tested and analyzed to determine their utility and feasibility in creating 3D biomimetic cardiac tissue.
  • Initial studies involved use of neonatal rat CMs and CFs, isolated by previously established procedures, encapsulated within a Collagen/Fibrin hydrogel and maintained within a cell suspension region 18 of a microfluidic device 10 for an extended l4-day culture period, while maintaining high cell survival. These experiments allowed for optimization of various parameters, such as viability, alignment, contraction, and cardiac marker expression. Specifically, varying cell ratios and compositions of hydrogels were studied to enhance tissue level alignment and viability of the cardiac tissue. FIG.
  • microfluidic devices 10 are tested with the microfluidic devices 10 to promote their optimization, with such variables including hydrogel composition, micropost shape, micropost length, widthwise post spacing (wPS), and lengthwise post spacing (IPS) (e.g., for the vertical posts 22 of FIGS. 2 and 3).
  • variables including hydrogel composition, micropost shape, micropost length, widthwise post spacing (wPS), and lengthwise post spacing (IPS) (e.g., for the vertical posts 22 of FIGS. 2 and 3).
  • FIGS. 8-11 provide phase contrast images for microfluidic devices 10 with different combinations of CM:CF cell ratios and geometries for the vertical posts 22.
  • FIG. 8 represents a 8:1 CM:CF cell ratio and a modified elliptical geometry for the vertical posts 22
  • FIG. 9 represents a 4:1 CM:CF cell ratio and a modified elliptical geometry for the vertical posts 22
  • FIG. 10 represents a 8:1 CM:CF cell ratio and a stretched hexagonal geometry for the vertical posts 22
  • FIG. 11 represents a 4:1 CM:CF cell ratio and a stretched hexagonal geometry for the vertical posts 22.
  • FIG. 12 is a still image obtained from a contraction video for the 4:1 cell ratio of CM:CF
  • FIG. 13 is a still image obtained from a contraction video for the 8:1 cell ratio of CM:CF.
  • FIGS. 14A, 15A, 16A, and 17A provide cytoskeletal staining images of rat cardiac tissue between the vertical posts 22 within microfluidic devices 10 according to four designs based on FIGS. 2 and 3, Design 1, Design 2, Design 3, and Design 4 respectively. All designs incorporate vertical posts 22 with a 500 pm length and a 100 pm width, with lengthwise post spacing (IPS) of 150 pm.
  • the geometry of the vertical posts 22 in Designs 1 and 2 (FIGS. 14A and 15 A) is modified elliptical, whereas the geometry of the vertical posts 22 in Designs 3 and 4 (FIGS. 16A and 17A) is stretched hexagonal.
  • the widthwise post spacing (wPS) of Designs 1 and 3 (FIGS.
  • FIGS. 14A and 16A is 200 um, and the widthwise post spacing (wPS) of Designs 2 and 4 (FIGS. 15A and 17A) is 150 um.
  • wPS widthwise post spacing
  • DAPI 4',6-diamidino-2-phenylindole
  • F-actin is represented in gray .
  • the inset images each represent a fast Fourier transform (FFT) of the actin stain.
  • FIGS. 14B, 15B, 16B, and 17B are color-inverted representations of the cytoskeletal staining images of 14A, 15A, 16A, and 17A, respectively.
  • FIGS. 18A and 19A provide Z-stacked images of immunostained cell matrices within a microfluidic device 10 according to Design 1 at 20X and 40X magnification, respectively, to demonstrate the 3D character of the tissue within the cell suspension region 18.
  • FIGS. 18B and 19B are color- inverted representations of the Z-stacked images of FIGS. 18A and 19 A, respectively.
  • FIG. 20 is a bar chart depicting calculated thickness of aligned tissues within microfluidic devices 10 according to Designs 1 to 4 (arranged sequentially from left to right), for three experiments with triplicate samples.
  • the specific dimensionalities of the microfluidic device 10 according to Design 1 were capable of maintaining thicker aligned tissue than microfluidic devices 10 according to Designs 2-4, demonstrating the effectiveness of Design 1 in mimicking anisotropy found in native heart tissue architecture.
  • Cell orientation analysis was performed through FFT analysis of actin- stained images of the cardiac tissue within a microfluidic device 10 as disclosed herein (e.g., according to Design 3). After binary thresholding of the FFT graph, lengths of the major and minor axes were measured. Then, using a previously established equation 1 , the orientation index was calculated for each microfluidic device 10 and averaged, to provide an orientation index value. An orientation index value of 0.566 was calculated for a microfluidic device 10 as disclosed herein.
  • the alignment of cardiac tissue within the cell suspension region 18 may be assessed through staining of the cytoskeletal marker, F-actin, and cell nuclei, DAPI, to quantify cellular alignment after two weeks in culture.
  • the samples may be fixed on Day 14 in 4% (v/v) paraformaldehyde (PF) solution in Dulbecco's phosphate-buffered saline (DPBS), and nuclei of the cells may be tagged with DAPI while F-actin cytoskeletal fibers may be stained with Alexa Fluor 488 phalloidin.
  • FIG. 21 is a plot of orientation of DAPI-stained nuclei of cells within microfluidic devices 10 according to Designs 1 to 4. Images were oriented so the alignment axis is 0°, as designated by tick marks along x-axis, with each bar representing a 10° deviation. As shown in FIG. 21, cells within microfluidic devices 10 according to Design 1 exhibit the highest degree of nuclei orientation.
  • cardiac tissue within the cell suspension region 18 may be assessed through staining of the cardiac markers sarcomeric alpha actinin and connexin 43.
  • the relative cardiac marker expression will determine optimal cell culture ratio and period of experimental culture. Additionally, videos of duration of thirty seconds can be recorded via phase microscopy of the cultured tissue to measure spontaneous beating at different time periods of culture.
  • Phase contrast and fluorescence images were acquired using Zeiss Axio Observer Zl equipped with Aptoome2 (Zeiss) and ZenPro software. Throughout cell culture period, samples were imaged every other day through phase contrast in 10X objective. Immunofluorescent images were taken at Day 14 using 10X, 20X, and 40X objectives and Z-stacked images were captured and reconstructed to form representative 3D images. Time-lapse imaging of the samples was completed on Days 8, 10, 12, and 14 to capture spontaneous contraction of the tissue. Movies were recorded at 10X objective for 30 seconds. [0089] To analyze cardiac tissue alignment, fluorescent images of samples, stained for F-actin and DAPI, captured on Day 14, were used. Images were processed using the NIH ImageJ and the orientations of the nuclei were extracted via the Analyze Particle plugin. For the analysis, two independent experiments were used, and each experiment had 2-3 technical replicates.
  • FIGS. 22-25 provide images of cardiac marker staining of rat cardiac tissue between vertical posts 22 for Design 1 to Design 4 of the microfluidic device 10 as disclosed herein.
  • the brightest spots are DAPI
  • medium gray areas are connexin 43
  • the darkest gray is sarcomeric alpha-actinin.
  • FIG. 26A is a 40X magnification image of the tissue within a microfluidic device 10 according to Design 1.
  • FIG. 26B is a color inverted copy of the image of FIG. 26A.
  • FIG. 26A shows striated sarcomeres and abundant expression of cardiac gap junctions.
  • FIG. 27 is a line graph plotting representative beating signals of the tissue for microfluidic devices 10 according to Design 1 to Design 4 (arranged sequentially from top to bottom). Tissue cultured within microfluidic devices 10 according to all four designs exhibited consistent spontaneous beating, demonstrating viability of the tissue.
  • FIGS. 28-31B represent items obtained from preliminary studies performed on a co culture of hiPSC-CMs and human CFs in hydrogel-encapsulated tissue within a cell suspension region 18 of a microfluidic device 10 as disclosed herein over a period of 6 days.
  • FIG. 28 is a phase contrast image, taken at day 6, of tissue between vertical posts 22 of the microfluidic device 10.
  • FIG. 29A is a cardiac marker stained image of the tissue surrounding a single vertical post 22 taken at day 1, with darker gray depicting live cells, and brighter spots depicting dead cells.
  • FIG. 29B is a color inverted copy of FIG. 29A.
  • FIG. 30 is a plot of a representative beating signal showing seven beats at regular intervals within a fifteen second period.
  • FIG. 31A is a cardiac marker stained image showing organization of actin fibers between vertical posts 22 of the microfluidic device 10, with an inset portion showing cardiac specific proteins sarcomeric alpha actinin and connexin 43.
  • FIG. 31B is a color inverted copy of FIG. 31A.
  • microfluidic devices 10 with the aforementioned dimensions, with widthwise post spacing (wPS) of 150 and 200 um (e.g., falling within a range of range of 150 pm - 250 pm), and geometries of the vertical posts 22 as elliptical and hexagonal. Additionally, the effect of microfluidic device 10 dimensions on tissue contraction and cardiac marker expression was analyzed. The microfluidic device 10 with optimized tissue thickness, contraction, and alignment was experimentally determined to be Design 1, with widthwise post spacing (wPS) of 200 um and elliptical geometry for the vertical posts 22.
  • wPS widthwise post spacing
  • the hydrogel-encapsulated cells within the microfluidic device 10 were fixed in 4% paraformaldehyde (PFA).
  • the microfluidic devices 10 were kept in an incubator (humidified, 37°C, and 5% C0 2 ) for 15 minutes. Afterward, the cells were rinsed with PBS-glycine 2X for 10 minutes of incubation each at room temperature. The final wash was with PBS-Tween-20 ((PBS -Polyoxyethylene (20) sorbitan monolaurate) (0.05% (v/v) Polyoxyethylene (20) sorbitan monolaurate in PBS) for 10 minutes at room temperature.
  • PBS-Tween-20 ((PBS -Polyoxyethylene (20) sorbitan monolaurate) (0.05% (v/v) Polyoxyethylene (20) sorbitan monolaurate in PBS) for 10 minutes at room temperature.
  • the cells were permeablized with 0.1% Triton-X-lOO for 30 minutes at room temperature.
  • blocking was then performed with 10% goat serum solution for one hour at room temperature.
  • the primary antibodies for sarcomeric alpha actinin and connexin 43 were diluted in 10% goat serum, and centrifuged at 14,000 RPM for 10 minutes. Then, these antibodies were applied to the samples and kept at 4°C overnight. The following day, the samples were washed with PBS-Tween-20 three times for 20 minutes each at room temperature. Then, the secondary antibodies were applied. After 30 minutes to 1 hour, the samples were washed with PBS-Tween-20 three times for 20 minutes each at room temperature.
  • Alexa Fluor488 Phalloidin and DAPI were added to the samples and left at 4°C overnight. Then the samples were washed with PBS-Tween-20 three times for 20 minutes each at room temperature. Finally, the samples were imaged using fluorescence microscopy (Zeiss Axio Observer Zl with the Zen Pro software suite) equipped with Apotome.2
  • hiPSC-CMs obtained from a commercial vendor (e.g., Cellular Dynamics International (CDI)) to be readily differentiated from hiPSCs based on standard protocols.
  • CDI Cellular Dynamics International
  • the frozen vial of hiPSC- CMs may be thawed into the CDI Plating Medium, and then the cells may be maintained in the CDI Maintenance Medium.
  • hiPSCs for differentiation of hiPSCs toward CMs, first hiPSCs are cultured in Geltrex coated plates.
  • 120 uL of Geltrex is thawed into 12 mL of DMEM/F-12K. Then 2 mL of this suspension is plated into each well of a 6-well plate. The plate is left in the incubator at 37°C for at least an hour. Then, the media is aspirated and the plate is ready to use for cell culture.
  • the E8 media for hiPSC culture is modified to contain 10 uM of Thiazovivin for 24 hours of culture, then the media is changed to E8 media.
  • E8 media is used for hiPSC culture, with media changes every day.
  • the CFs (either rat- or human-derived) need to be dissociated from their culture flask.
  • aspirate media wash the vessel with IX DPBS, then add Trypsin IX and incubate at 37°C for ⁇ 5 minutes until the cells begin to round up.
  • wash the vessel with complete DMEM (DMEM +l0%FBS + 1% Pen/strep + 1% L-glutamine).
  • DMEM +l0%FBS + 1% Pen/strep + 1% L-glutamine Then collect the cell suspension and count via hemocytometer to determine volume resuspension for desired cell encapsulation density.
  • the cells are centrifuged at 200g for 5 minutes.
  • the supernatant is aspirated, then the cell pellet is resuspended in predetermined volume of complete DMEM.
  • the CMs and the CFs are mixed together at the desired ratio (4:1), then mixed with 2 mg/mL fibrinogen, thrombin at 1 U/mg of fibrinogen, and 1 mg/mL collagen.
  • microfluidic devices 10 have demonstrated their utility in establishing a 3D cardiac tissue of both primary animal- and stem cell-derived CMs.
  • FIGS. 32A-34B represent items obtained from additional studies performed on a co culture of hiPSC-CMs and human embryonic stem cell-derived cardiomyocytes (hESC-CMs), together referred to as human pluripotent stem cells (hPSCs), in hydrogel-encapsulated tissue within the cell suspension region 18 of the microfluidic device 10 as disclosed herein.
  • hPSC-CMs were encapsulated with CFs in a 3D biomimetic hydrogel, and the celkhydrogel suspension was injected into the microfluidic device 10.
  • FIGS. 32A-32C provide a characterization of the cardiac cell population from differentiation of hiPSCs.
  • FIG. 32A is an immunofluorescent stained image of sarcomeric alpha actinin (a -actinin, lighter gray) and vimentin (medium gray) to identify populations of cardiomyocytes and cardiac fibroblasts, respectively.
  • FIG. 32B is an immunofluorescent stained image of sarcomeric alpha actinin (lighter gray) and connexin 43 (CX43, medium gray) of hiPSC-CM population.
  • FIG. 32C is a magnified image of the immunofluorescent stained image of FIG. 32B.
  • a device without the staggered elliptical microposts within the channel (referred to as“no posts”), was used to house the cardiac tissue for the same period, and the resulting tissue was compared to that formed in the proposed microfluidic device 10. It was found that the cardiac tissues within the microfluidic device 10 with the vertical posts 22 were more aligned, elongated, and exhibited a high degree of maturation, through gene and protein level expression, than in the devices without the posts.
  • the culture of cells within a 3D hydrogel around the precisely spaced elliptical vertical posts 22 enabled the creation of a highly aligned cardiac tissue, derived from human stem cells, with a maturation state more physiologically relevant to the human myocardium than hPSC-CMs cultured in typical monolayer format.
  • FIGS. 33A-33C provide an assessment of the alignment of hiPSC-derived cardiac tissues formed within devices with and without the vertical posts 22.
  • FIG. 33A is an Actin (medium gray) stained image of cardiac tissues within the microfluidic device 10 including vertical posts 22.
  • FIG. 33B is an Actin (medium gray) stained image of cardiac tissues within devices without posts.
  • FIG. 33C is a plot quantifying cell alignment around an alignment axis, demonstrating a higher proportion of cells aligned along the alignment axis (i.e. at 0°) in cardiac tissues formed in the microfluidic device 10 with vertical posts 22 than in devices without posts.
  • FIGS. 34A-34C provide images of expression of cardiac markers of hiPSC-derived cardiac tissues inside the microfluidic device 10 and devices without posts.
  • FIG. 34A is an immunofluorescent stained image of sarcomeric alpha actinin (lighter gray) and CX43 (medium gray) of cardiac tissues formed after 2 weeks in the microfluidic device 10 with vertical posts 22.
  • FIG. 34B is an immunofluorescent stained image of sarcomeric alpha actinin (lighter gray) and CX43 (medium gray) of cardiac tissues formed after 2 weeks in devices without posts.
  • FIG. 34C is a magnified image of the immunofluorescent stained image of FIG. 34A.
  • cardiac cells were used to show the success of the platform, cells of other types such as neural, embryonic stem, or skeletal cells could be used in microfluidic devices 10 as disclosed herein. As will be recognized by one skilled in the art, culture details may vary depending on the types of cells used.
  • Embodiments disclosed herein may provide one or more of the following technical benefits.
  • Microfluidic devices 10 disclosed herein integrate highly organized microposts (e.g., the vertical posts 22) with a cell suspension region 18 to induce 3D cellular and tissue-level alignment similar, for instance, to the architecture of the native human myocardium in a microscale platform.
  • Microfluidic devices 10 disclosed herein also enable enhanced sample collection (i.e., cells) from the cell suspension region 18 by providing separate sample extraction ports 40, 42 downstream of two lateral portions of the cell suspension region 18 to enable performance downstream genetic analyses.
  • microfluidic devices 10 disclosed herein enable sufficient collection of media samples from the first fluid channel 44 and the second fluid channel 46 for analysis on the secreted cytokines/proteins using standard assays such as ELISA or LCMS. Moreover, microfluidic devices 10 disclosed herein provide the ability to create specific injury and/or disease models through manipulation of the precisely controlled factors within the tissue microenvironment.
  • This platform allows for specific and directed application of insults, such as drugs or environmental factors, on the encapsulated tissue (e.g., cardiac, brain, or skeletal tissue). Additionally, the platform allows for continuous, real-time monitoring of the cardiac tissue in both the healthy and injured/diseased state.
  • insults such as drugs or environmental factors

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Abstract

L'invention concerne un dispositif microfluidique qui incorpore une co-culture hautement organisée de cellules vivantes à l'intérieur d'une plate-forme micro-modifiée, par lequel l'architecture et les constituants cellulaires d'un organe ou d'un autre environnement de tissu natif peuvent être modélisés sur une longue période de culture pour des études biologiques et/ou pharmacologiques. Des montants verticaux (par exemple, des micromontants) peuvent être utilisés pour induire l'alignement de tissus encapsulés dans un hydrogel dans une région de suspension cellulaire d'un dispositif microfluidique. Un tel dispositif peut compléter des études animales en récapitulant les caractéristiques physiopathologiques de la maladie. Lorsqu'elle est peuplée avec des cellules cardiaques, un tel dispositif fournit une forme tridimensionnelle (3D) un modèle de tissu cardiaque humain biomimétique qui permet l'étude d'événements physiopathologiques impliqués dans la transition de tissus cardiaques sains à malades, pour mieux informer des stratégies thérapeutiques et des résultats fonctionnels dans des thérapies à base cardiaque. D'autres types de cellules peuvent être utilisés dans certains modes de réalisation. L'invention concerne également des procédés de fabrication et d'utilisation de tels dispositifs microfluidiques.
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CN117836051B (zh) * 2021-07-28 2025-03-25 北加州大学药学院 多层微流控系统及方法
US12303891B2 (en) 2021-07-28 2025-05-20 California Northstate College Of Pharmacy, Llc Emulating vascular cellular physiologies
JP7784760B2 (ja) 2021-07-28 2025-12-12 カリフォルニア ノースステイト カレッジ オブ ファーマシー， エルエルシー 多層マイクロ流体システムおよび方法
WO2025125848A1 (fr) * 2023-12-15 2025-06-19 Université De Technologie De Compiègne Biopuce pour culture cellulaire

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