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WO2023009645A1 - Système microphysiologique in vitro de système vasculaire vasoactif - Google Patents

Système microphysiologique in vitro de système vasculaire vasoactif Download PDF

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Publication number
WO2023009645A1
WO2023009645A1 PCT/US2022/038551 US2022038551W WO2023009645A1 WO 2023009645 A1 WO2023009645 A1 WO 2023009645A1 US 2022038551 W US2022038551 W US 2022038551W WO 2023009645 A1 WO2023009645 A1 WO 2023009645A1
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Prior art keywords
microvessels
living
cells
drug
microfluidic channel
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PCT/US2022/038551
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English (en)
Inventor
Christopher C.W. HUGHES
R. Hugh F. BENDER
Duc T.T. PHAN
Abraham P. Lee
Steven C. George
G. Wesley Hatfield
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Aracari Biosciences Inc
University of California Berkeley
University of California San Diego UCSD
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Aracari Biosciences Inc
University of California Berkeley
University of California San Diego UCSD
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Publication of WO2023009645A1 publication Critical patent/WO2023009645A1/fr
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    • 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
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • 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
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • 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
    • C12N2510/00—Genetically modified 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
    • C12N2513/00—3D culture
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/56—Fibrin; Thrombin
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to in vitro microtissues systems and production methods thereof; more particularly, to microtissues constructed on a microfabricated platform and perfused with living microvessels.
  • the present invention further relates to biomedical engineering, tissue engineering, cardiovascular drug development, drug delivery, drug screening, disease modeling, clinical diagnostics, microfluidic technology, and microphysiological systems.
  • microfabrication technology has led to the creation of precise microchannels on non-biological substrates (e.g., silicon or polydimethyl siloxane, PDMS) 1 ⁇ 2 , or within biological substrates such as collagen 3 .
  • non-biological substrates e.g., silicon or polydimethyl siloxane, PDMS
  • PDMS polydimethyl siloxane
  • these approaches offer the distinct advantage of introducing advection as a mechanism of transport, even when “endothelialized,” the channels are not living microvessels.
  • this approach may assist the creation of larger engineered tissues, they are of less benefit in understanding in vivo biological functions such as angiogenesis, cell migration, cell differentiation, and ischemia.
  • Interstitial fluid flow can markedly impact extracellular gradients of solutes, enhance transport of nutrients and waste, and significantly impact the development of both lymphatic and blood capillaries 46 .
  • These recent studies, as well as others 79 highlight the ability to generate living microvessels in 3D and also demonstrate that these living microvessels can become functional upon implantation.
  • vasoactive network for testing new drugs.
  • a major problem when introducing a new drug to clinical trials is the occurrence of unwanted effects on vascular tone (i.e., vasoactivity) and blood pressure.
  • vascular tone i.e., vasoactivity
  • blood pressure i.e., blood pressure
  • hypertension and hypotension can have immediate and long-term life-threatening effects on patients and are almost always disqualifying for further development of the drug.
  • hypertension is increasing in western societies, and therefore, so too is the need for vasoactive drugs.
  • Some embodiments of the invention are set forth in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.
  • the present invention features a vasoactive network platform (the VMO-VA).
  • the platform may contain a plurality of individual device units, each unit with perivascular cells (e.g., smooth muscle cells, myofibroblasts, pericytes, etc.) that wrap around fully- functional human microvessels to create a vascular network perfused with a blood substitute.
  • perivascular cells e.g., smooth muscle cells, myofibroblasts, pericytes, etc.
  • the cells comprising the blood vessels including endothelial cells and the contractile perivascular cells, respond to both vasodilator and vasoconstrictor drugs in the same way that blood vessels do in the body.
  • the platform can be used to test novel drugs for wanted or unwanted vasoactive effects on the blood vessels.
  • the platform can also be used to test responses to an increase in blood pressure.
  • the platform may be compact, as small as palm-sized.
  • One of the unique and inventive technical features of the present invention is the use of perivascular cells to support and form an ex vivo vascular network. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides an ex vivo human vascular network that may be used to assess the vasoactivity of new drugs. None of the presently known prior references or work has the unique technical feature of the present invention. Previous work disclosed in U.S. Patent No. 9,810,685 describes a perfusable vascular network but does not have the capacity for vasoactivity.
  • perivascular cells will support the formation of a lumenized vascular network needed to test the vasoactivity of drugs without modification in soluble factors and extracellular matrix components.
  • lumenized vessels do not form well when certain types of smooth muscle cells are used (i.e., primary human umbilical artery smooth muscle cells, primary human aortic smooth muscle cells, and immortalized human aortic smooth muscle cells).
  • smooth muscle cells i.e., primary human umbilical artery smooth muscle cells, primary human aortic smooth muscle cells, and immortalized human aortic smooth muscle cells.
  • stromal cells support formation of the lumenized vascular network, but they do not respond to vasoreactive stimuli under low shear stress.
  • the secretion of extracellular matrix components may help promote vessel lumen formation.
  • Another inventive technical feature of the present invention is the combination of cell type and vascular shear stress applied to the microvessels.
  • the present invention may use low vascular shear stress in combination with contractile perivascular cells.
  • a low vascular shear stress does not affect the phenotype of the cells.
  • Low vascular shear stress may be less than about 5 dyn/cm 2 . None of the presently known prior references or work has the unique technical feature of the present invention.
  • this inventive technical feature of the present invention contributed to a surprising result. For example, the body does not have the combination of low vascular shear stress with contractile perivascular cells.
  • the shear stress applied to the vascular network may affect the phenotype of the cells.
  • a high vascular shear stress may be used in combination with cells that are not contractile.
  • high vascular shear stress caused a change in phenotypes of cells.
  • cells that are not originally contractile may adopt this phenotype under high vascular shear stress.
  • High vascular shear stress may be greater than about 5 dyn/cm 2 .
  • the present invention may additionally feature a tissue receiving device comprising one or more units.
  • Each unit may comprise a first microfabricated microfluidic channel and a second microfabricated microfluidic channel formed within a non-biological supportive material, and one or more microtissue compartments formed within the non-biological supportive material.
  • at least one of the one or more microtissue compartments comprises one or more living microvessels.
  • the one or more living microvessels includes a first living microvessel that connects to the first microfabricated microfluidic channel and the second microfabricated microfluidic channel, the first living microvessel having a lumen and running from the first microfabricated microfluidic channel, through at least one microtissue compartment (105), and to the second microfabricated microfluidic channel.
  • the present invention may further feature a method of vascularizing a spheroid (or organoid; e.g., a patient-derived spheroid or organoid).
  • the method may comprise adding a first media to a tissue receiving device as described herein, and allowing the one or more living microvessels (120) to form over a period of time.
  • the first media is then aspirated out and a spheroid is added to at least one the one or more loading ports disposed on the one or more microtissue compartments (105).
  • a second media may then be added to the device.
  • FIG. 1 shows one embodiment of a fabrication scheme for in vitro metabolically active microtissues perfused with human microvessel bed.
  • FIG. 2A shows a cut-away view of microtissue compartment highlighting the communication ports that will allow sprouting microvessels to penetrate the microtissue from either arteriole or venule microfluidic channels, as well as the porous lower parylene layer for lymph drainage.
  • FIG. 2B shows a top view highlighting microfluidic channels and communication ports.
  • FIG. 2C shows venule and arteriole microfluidic channels coated with collagen (I or IV) or fibronectin, seeded with endothelial cells (black dashes) which sprout into the microfluidic channels as microvessel through the ports to deliver nutrients to the metabolizing microtissue.
  • collagen I or IV
  • fibronectin endothelial cells
  • FIG. 3 shows another embodiment of a fabrication scheme for in vitro metabolically active microtissues perfused with human microvessels.
  • FIGs. 4A and 4B show two examples of fabrication schemes/arrangements for high throughput in vitro perfused human microvessels.
  • FIG. 5 shows some dimensions which define the A-V system connected by the microtissues.
  • FIG. 6 shows another embodiment of a fabrication arrangement.
  • FIGs. 7A, 7B, and 7C show embodiments in which the resistance to fluid flow in the microfluidic channels and the interstitial flow (flow through the tissue but not through the microvessels) through the microtissues can be manipulated to be predominantly longitudinal (FIG. 7A and 7B) or lateral (FIG. 7C).
  • FIG. 8 shows another embodiment in which the microtissue “channel” is shaped like a diamond to provide separation (diffusion and/or convection) of this tissue from neighboring tissues.
  • FIG. 9A shows an embodiment of the microfluidic platform (100A) that has a single unit and four reservoirs (e.g., V1, V2, V3, and V4).
  • Three tissue compartments e.g., 1.0 x1.0 x0.1mm) constitute 1 unit.
  • Different levels of medium in the four reservoirs drive flow and can generate high shear stress to stimulate perivascular cells turning into smooth muscle cells.
  • FIG. 9B shows another embodiment of the microfluidic platform (100B) that is configured for a 96-well plate footprint and has 12 units/plate. This embodiment can only generate low shear stress, and thus requires a specific type of smooth muscle cell (i.e. human pulmonary smooth muscle cells).
  • FIG. 9C shows a compact microfluidic design for 96-well plate format.
  • FIG. 9D shows a single tissue compartment flanked by a microfluidic “arteriole” and “venule.”
  • FIG. 9E shows a vascular network in the tissue compartment. Endothelial cells (EC) are expressing EGFP.
  • FIG. 9F shows 70kDa Rhodamine dextran flowing through the upper “arteriole”, through the capillary network, and out through the low pressure “venule” at the bottom.
  • FIG. 9G shows an EC-lined microfluidic channel (*) and sprouting capillaries.
  • FIG. 9H shows a collagen IV- rich basement membrane surrounds a vessel.
  • FIG. 91 shows a vessel (mCherry) wrapped by a pericyte (YFP) supplies nutrients to surrounding cells (not visualized).
  • FIG. 9J shows leakage of 150kDa FITC-dextran is minimal over 90 mins. Calculated permeability coefficient of 1.2 x 10 ® cm/s is consistent with capillary permeability in vivo.
  • FIG. 10A shows a representative image of a mature, interconnected vascular network supported by HPSMC in the VMO-VA platform.
  • FIG. 10B shows representative images of 70-kDa fluorescent dextran perfusion in the stromal cells-VMO and HPSMC-VMO-VA platforms.
  • FIG. 11 A shows Z-projection of confocal images of a-SMA and transgelin immunostaining that shows that a-SMA+/transgelin+ cells tightly wrapped around a vertical segment of lumenized vessel.
  • FIG. 11 B shows orthogonal views revealed a-SMA+/transgelin+ (arrows heads) were in close contact with the basal side of the vessel (small arrows). Asterisk (*) indicates vessel lumen.
  • FIG. 12 shows vasoactive drugs and their mechanism of action.
  • Two vasoconstrictors and two vasodilators were tested against the VMO-VA platform.
  • the vasodilators nitroprusside and nifedipine only target SMC.
  • FIGs. 13A and 13B show dose-response curves of %Activation of L-NMMA (FIG. 13A) and phenylephrine (FIG. 13B) at 4 hours post-drug treatment.
  • FIGs. 13C and 13D show representative images of strongly constricted vessels (circleed) in response to L-NMMA (FIG. 13C) and phenylephrine (FIG. 13D).
  • FIGs. 14A and 14B show a dose-response curve of %Activation of nifedipine (FIG. 14A) and nitoprusside (FIG. 14B) at 4 hours post-drug treatment.
  • FIGs. 14C and 14D show representative images of obviously dilated vessels (circled) in response to nifedipine (FIG. 14C) and nitoprusside (FIG. 14D).
  • FIGs. 15A-15D show a time course study at various drug doses for: L-NMMA (FIG. 15A); phenylephrine (FIG. 15B); nitroprusside (FIG. 15C); and nifedipine (FIG. 15D) showed that vasoactivity was sustained for all except phenylephrine.
  • FIG. 15A at the end of the time course the lines in descending order are 0 nM, 7 nM, 14 nM, 70 nM, and 700 nM.
  • FIG. 15B at the end of the time course the lines in descending order are 8800 nM, 177 nM, 88 nM, 880 nM, and 0 nM.
  • the lines in descending order are 150 nM, 15 nM, 1.5 nM, and 0 nM.
  • the lines in descending order are 640 nM, 64 nM, 12.8 nM, 6.4 nM, and 0 nM.
  • FIG. 16 shows representative images of endothelial cells co-cultured with stromal cells (top panel), AO-SMC (middle panel), and UA-SMC (bottom panel).
  • FIG. 17 shows a non-limiting example of a device comprising one or more loading ports that are able to receive a tissue (e.g., a spheroid; e.g., an organoid).
  • a tissue e.g., a spheroid; e.g., an organoid
  • FIGs. 18A and 18B show non-limiting examples of methods that may be used to add a tissue to a device comprising one or more loading ports.
  • FIG. 19 shows an alternative embodiment of a device comprising one or more loading ports that are able to receive a tissue (e.g., a spheroid; e.g., an organoid).
  • a tissue e.g., a spheroid; e.g., an organoid
  • FIG. 20 shows a non-limiting example of a method utilizing the device shown in FIG. 19.
  • microvessels or “living microvessels” include arterioles, capillaries, venules, and lymphatics vessels. These living microvessels produced by the various embodiments connect the microfluidic channels to the microtissue. These microvessels are formed within the “pores” structures/channels located within the microfluidic channels. Microvessels described herein are metabolically active.
  • microfluidic channels refer to the disclosed “arteriole” or “venuole” supplying channels, with respect to supplying or removing material from the microtissue compartment. “Arterioles” supply nutrients/fluid etc. to the microtissue; whilst “venuoles” remove nutrients/fluid from the microtissue. These microfluidic channels are created by microfabrication technology and are not considered “3-D metabolically active” or living vessels.
  • microtissue compartment refers to a location where cells are grown. This term includes embodiments where microtissues are grown in channels rather than in closed and isolated compartments.
  • the term “stimulus” refers to a condition that can be induced both mechanically (e.g., with interstitial flow and pressure) or chemically (e.g., with growth factors (e.g., VEGF), pH, or hypoxia) or any combination thereof, which is applied to the microfluidic channel (and/or cells thereof).
  • the stimulus can also be generated/produced by the cells within the microfluidic channels themselves or from the microtissue channels (and/or cells thereof).
  • the term “fluid” refers to a liquid that is able to flow.
  • the liquid can be blood, saline, buffer, culture media or any other solvent or media, whether the liquid is native or artificially produced.
  • the ability of the microvessels and microfluidic channels to be able to deliver fluid can be assessed using various methods known to those of skill in the art, including but not limited to, imaging fluorescent molecules (e.g., different molecular weight fluorescent dextrans) or fluorescent microcarrier beads (diameter less than the diameter of the pore and microvessel) that have been initially placed in the microfluidic channel.
  • abnormal cells refers to cells that are considered disease-free, whether they are obtained from disease-free or asymptomatic human individuals or animals.
  • diseased or abnormal in the context of “diseased/abnormal cells” refers to cells obtained from human individuals or animals who suffer from an illness or disease known to those of skill in the art.
  • drug refers to any known compound or composition, ora combination thereof, that is used to treat any disease. Such drugs are well known to those of skill in the art. The term also refers to compounds or compositions which are considered candidate “drugs.” In some embodiments, devices described herein may be used to test potential side effects (e.g., vasodilation or vasoconstriction) of candidate drugs.
  • perivascular cells refers to cells that are situated around a blood vessel. Perivascular cells can be isolated from multiple tissues in the body and play a role in tissue repair, vascular homeostasis as well as angiogenesis. Perivascular cells include mainly two types of cells: vascular smooth muscle cells (SMCs) and pericytes. Examples of perivascular cells include, but are not limited to, any cell type that has the ability to wrap around the vessels and change the vessel diameter by constricting or dilating the vessel wall, such as a myofibroblast, mesenchymal stem cell, pericyte, smooth muscle cell, or other stromal or fibroblast-like cells.
  • the approaches described herein to create a 3D perfused human microvessel bed combines 3D cell culture and microfabrication technology and includes not only the flexibility for high-throughput design drug screening but also real-time monitoring.
  • the overall strategy is biology-directed and inspired by the in vivo steps of angiogenesis and vasculogenesis.
  • a minimal architecture i.e., matrix, angiogenic stimuli
  • the endothelial cells and contractile perivascular cells are allowed to create a network of microvessels to meet metabolic needs.
  • the present invention describes a method for creating a metabolically active microtissue that receives nutrients and eliminates waste products through a living microvessel network.
  • the long-term survival e.g., weeks to months
  • the present invention describes a method for creating a direct fluidic connection between living microvessels within the device and microfluidic channels (i.e., A-V “arterio-venous” channels) within the device allowing the flow of fluid between the microfluidic channels and the microvessels.
  • the microvessels are within the metabolically active tissue and can thus be perfused to deliver nutrients and remove waste products by convection in addition to diffusion.
  • Templates for the microfluidic device may comprise materials known to those of skill in the microfabrication, such as, but not limited to, PDMS, glass and/or other polymer materials. Standard methods are used to etch or mold such micro fluidic channels into these templated materials.
  • microtissue(s) (110) resides in the microtissue compartment (105).
  • the newly developed living microvessels (120), growing within the microtissue (110), are able to deliver fluid from said microfluidic channels (140), through the ports, and into said microtissues (110) growing within the microtissue compartment (105).
  • This fluid e.g., blood, cell culture media
  • Said fluid may also contain stimulants and reagents to test the response of the microvessels, e.g., a drug.
  • an article comprising a supportive structure, one or more microfluidic channels (140), one or more microtissue compartments (105), and one or more microvessels (120), wherein the microvessels (120) connect said microfluidic channels (140) and microtissue (110) and perfuse said microtissue (110), thereby allowing delivery of fluid or nutrients from the microfluidic channels (140) to the microtissues (110).
  • a process for creating a 3D metabolically active network of living microvessels (120) may comprise preparing a template comprising a plurality of microfluidic and microtissue channels (140), and providing a stimulus to said microfluidic channels (140), whereby the stimulus creates a 3D metabolically active network of living microvessels (120).
  • the microvessels (120) connect the microfluidic and microtissue channels (140) and deliver fluid between said channels (140) and/or the microvessels (120) are formed within the microtissue (110).
  • the present invention features a microphysiological system (100) for testing the vasoactivity of a drug.
  • the system (100) may comprise a plurality of individual devices.
  • the individual devices may comprise individual tissues.
  • each tissue comprises one or more perfusable microvessels (120) and contractile perivascular cells (130) that wrap around the microvessels (120).
  • perivascular cells include, but are not limited to, any cell type that has the ability to wrap around the vessels and change the vessel diameter by constricting or dilating the vessel wall, such as a myofibroblast, mesenchymal stem cell, pericyte, smooth muscle cell, or other stromal or fibroblast-like cells.
  • one contractile perivascular cell type is used.
  • one or more contractile perivascular cell types are used.
  • the system (100) is used to test the vasoactivity of a drug.
  • the perivascular cells may react to the drug by dilating or constricting the one or more microvessels (120).
  • the present invention may feature a microphysiological device (100A) for testing the vasoactivity of a drug.
  • the device (100) comprises one or more compartments (105), a microfluidic channel disposed on opposing ends of each compartment such that the one or more compartments (105) are in fluid connection with each other, and a blood substitute perfused through the device (100).
  • each compartment (105) comprises a plurality of individual tissues.
  • each tissue comprises one or more microvessels (120) with contractile perivascular cells (130) that wrap around the microvessels (120).
  • the contractile perivascular cells (130) may be transduced with lentivirus expressing fluorescent proteins.
  • the one or more microvessels (120) have a diameter of about 5 pm to 500 pm.
  • the present invention features a method for forming microvessels in the systems and devices described herein.
  • the method may comprise adding contractile perivascular cells (130) and endothelial cells (135) to a compartment and inducing a stimulus to the endothelial cells (135).
  • the stimulus causes endothelial cells (135) to form the microvessels (120).
  • the stimulus applied to the endothelial cells may be induced mechanically, chemically, ora combination thereof. Examples of ways to induce the stimulus include, but are not limited to, interstitial flow, pressure, growth factors, pH, hypoxia, or a combination thereof.
  • the present invention features a method of forming a vascular network in vitro for testing vasoactivity of a drug.
  • the method may comprise placing contractile perivascular cells (130) and endothelial cells (135) in a compartment (105) and generating one or more perfusable microvessels (120) comprising contractile perivascular cells (130) wrapped around at least a portion of each perfusable microvessel (120) in response to a first stimulus.
  • the first stimulus comprises a pro-angiogenic stimulus.
  • the method further comprises adding a second stimulus to modify the vasoactivity of the one or more perfusable microvessels (120).
  • adding the second stimulus causes the one or more perfusable microvessels (120) constrict. In some embodiments, adding the second stimulus causes the one or more perfusable microvessels (120) dilate. In other embodiments, adding the second stimulus does not modify the vasoactivity of the one or more perfusable microvessels (120).
  • the present invention features a method for wrapping contractile perivascular cells (130) around microvessels (120).
  • the method may comprise adding contractile perivascular cells (130) and endothelial cells (135) to a compartment and subsequently inducing a stimulus to the contractile perivascular cells (130).
  • the stimulus causes the contractile perivascular cells (130) to wrap around the microvessels (120).
  • the contractile perivascular cells (130) come closer to the microvessels (120), then pull themselves or wrap around a portion of the microvessels (120).
  • the contractile perivascular cells (130) are not attached to the microvessels.
  • the contractile perivascular cells (130) may form a basement membrane around the microvessels (120).
  • the basement membrane may be comprised of extracellular matrix components.
  • the basement membrane is shared between the endothelial cells and the contractile perivascular cells.
  • the contractile perivascular cells are recruited to close proximity with the microvessels in response to paracrine signals from the endothelial cells.
  • endothelial cells and the contractile perivascular cells synthesize basement membrane proteins, which sandwich between the contractile perivascular cell bodies and the outer (basal) side of the microvessels. This basement membrane covers the entire side of the microvessels, and the contractile perivascular cell bodies lay flat along the basement membrane.
  • the stimulus applied to the contractile perivascular cells may be induced mechanically, chemically, ora combination thereof.
  • Examples of ways to induce the stimulus include, but are not limited to, interstitial flow, pressure, growth factors, drugs, pH, hypoxia, or a combination thereof.
  • the method may comprise inducing a stimulus to both the endothelial cells and the contractile perivascular cells.
  • the contractile perivascular cells (130) may be myofibroblasts, smooth muscle cells, pericytes, mesenchymal stem cells, fibroblast-like cells, or a combination thereof.
  • human contractile perivascular cells (130) and endothelial cells (135) are allowed to form a microvessel network within a microtissue (110) in response to normal or pathologic angiogenic stimuli.
  • the angiogenic stimuli initially may be some added growth factors in the fluid in the A-V (arteriole-venule) microfluidic channels (140), but eventually, the cells in the microtissue compartment (105) may produce the angiogenic growth factors/stimuli.
  • the stimuli may be present simultaneously in the A-V microfluidic channels (140) and microtissue compartment (105).
  • the stimuli may only be present in the microtissue compartment (105).
  • the microtissue compartment (105) is a closed and controlled environment in which nutrients and waste only enter and exit from a controlled number of openings (ports) in the adjacent fluid-filled A-V channels (140).
  • the flow of fluid is initially through the interstitial space, but as the microvessel network forms, the flow of fluid can divert to the living microvessels (120) that are formed between the microfluidic and microtissue (110) and within the microtissue (110) itself.
  • the stimuli could be added to the ports.
  • the angiogenic stimuli are biologically-induced and can be both mechanical (interstitial flow and pressure) and chemical (e.g., VEGF, pH, or hypoxia) in nature.
  • the microtissue compartment is comprised of either fibrin, type I collagen, or other biomimetic matrices (synthetic or naturally occurring) as well as human contractile perivascular cells (e.g., myofibroblasts, smooth muscle cells, pericytes, mesenchymal stem cells, fibroblast-like cells, or a combination thereof), which is necessary for sustained lumen formation, and, in this application, for facilitating a metabolic deficit.
  • perivascular cells e.g., myofibroblasts, smooth muscle cells, pericytes, mesenchymal stem cells, fibroblast-like cells, or a combination thereof
  • a physiological pressure gradient within the microtissue compartment can be provided to initially induce limited nutrient fluid flow through the microtissue (110).
  • the microvessels (120) sprout out from the microfluidic channels (140) and grow towards the angiogenic stimulus, they eventually meet, anastomose, and deliver nutrients to the metabolizing microtissue (110).
  • this system (100) mimics in vivo angiogenesis and vasculogenesis. Additional details on the cells, matrix, and microfabrication are discussed below.
  • microfluidic channels (140) and microtissue environments can be carefully controlled within physiologic ranges by manipulating either the inlet or outlet pressures and/or the design of the microfluidic network. Such manipulation of flow pressures can also be used as a stimulus to the microfluidic channel cells to produce the microvessels (120).
  • the new microvessels are formed within the microtissue (110) and then connect to the microfluidic channels (140).
  • the newly formed microvessels grow/sprout from the microfluidic channels (140), grow into the microtissue (110), and connect with the microvessels that are growing in from the other side.
  • a combination of both of the former two microvessel growth paths can occur.
  • contractile perivascular cells may be used in combination with a low vascular shear stress.
  • a low vascular shear stress may be less than about 5 dyn/cm 2 .
  • An example of cells that may be used in combination with low vascular shear stress include human pulmonary smooth muscle cells.
  • a low vascular shear stress may be less than about 0.5 dyn/cm 2 , or less than about 1.0 dyn/cm 2 , or less than about 1.5 dyn/cm 2 , or less than about 2.0 dyn/cm 2 , or less than about 2.5 dyn/cm 2 , or less than about 3.0 dyn/cm 2 , or less than about 3.5 dyn/cm 2 , or less than about 4.0 dyn/cm 2 , or less than about 4.5 dyn/cm 2 , or less than about 5.0 dyn/cm 2 .
  • a high vascular shear stress may be used in combination with cells that are not contractile.
  • cell types that may be used in combination with a high vascular shear stress include but are not limited to fibroblast-like cells, mesenchymal stem cells, myofibroblasts, pericytes, or a combination thereof.
  • a high vascular shear stress may be greater than about 5 dyn/cm 2 .
  • a high vascular shear stress may cause a change in the phenotype of the cells. For example, cells that are not originally contractile may adopt this phenotype under high vascular shear stress.
  • a high vascular shear stress may be greater than about 5.0 dyn/cm 2 , or greater than about 6.0 dyn/cm 2 , or greater than about 7.0 dyn/cm 2 , or greater than about 8.0 dyn/cm 2 , or greater than about 9.0 dyn/cm 2 , or greater than about 10.0 dyn/cm 2 , or greater than about 11.0 dyn/cm 2 , or greater than about 12.0 dyn/cm 2 , or greater than about 13.0 dyn/cm 2 , or greater than about 14.0 dyn/cm 2 , or greater than about 15.0 dyn/cm 2 .
  • the system (100) may further comprise a medium.
  • the medium may be blood or a blood substitute.
  • the blood substitute include modified cell culture media with relevant soluble components of human blood plasma.
  • the contractile perivascular cells (130) may or may not be transduced with lentivirus expressing fluorescent proteins. Without wishing to limit the present invention to any theory or mechanism, fluorescently labeling the perivascular cells allows for imaging of the microvessels (120), which may be used for measuring the vasoactivity of tested drugs.
  • the one or more microvessels (120) have a diameter of about 5 pm and up to about 500 pm.
  • the present invention features methods for testing the vasoactivity of a drug using the systems and devices described herein.
  • the method comprises measuring a diameter of one or more microvessels (120) using a fluorescent dye, adding the drug to a medium, flowing the medium containing the drug through the system (100), re-introducing a fluorescent dye through the system (100) with the drug, and measuring the diameter of one or more microvessels (120) over time.
  • the medium may be blood or a blood substitute.
  • one or more microvessels (120) constrict in the presence of the drug. In other embodiments, one or more microvessels (120) dilate in the presence of the drug. In one embodiment, one or more microvessels (120) do not change in diameter (i.e., do not dilate or constrict) in the presence of the drug. In yet another embodiment, the diameter of one or more microvessels (120) is measured by fluorescent imaging of one or more microvessels (120). In further embodiments, the medium flows through the system (100) for a period of time. In other embodiments, a time period for testing drugs in the system (100) is about 1 h to 24 h. In further embodiments, the system (100) described herein may be maintained for a period of time up to about 14 days.
  • the vasoactivity of a drug may be measured by the flow rate or flow velocity of the medium in one or more microvessels (120) in the system (100).
  • the method comprises adding medium to the system (100), flowing the medium through the system (100), measuring an initial flow rate or flow velocity of the medium in one or more microvessels (120), adding the drug to the medium, flowing the medium containing the drug through the system (100), and measuring a final flow rate or flow velocity in one or more microvessels (120) of the drug-containing medium after a period of time.
  • tracer molecules or microbeads may be added to the medium.
  • flow may be measured by introducing microbeads into the system (100) and measuring their velocity in one or more microvessels (120) in the system (100) or measuring the time it takes for the microbeads to leave the system (100) in a given vascular flow path.
  • tracer molecules may be introduced into the system (100). Methods to track the tracer molecules in the system (100) include, but are not limited to, particle tracking velocimetry, laser speckle imaging, or other techniques that may include fluorescent imaging.
  • one or more microvessels (120) constrict in the presence of the drug. In other embodiments, one or more microvessels (120) dilate in the presence of the drug. In one embodiment, one or more microvessels (120) do not change in diameter (i.e., do not dilate or constrict) in the presence of the drug. In yet another embodiment, the diameter of one or more microvessels (120) is measured by fluorescent imaging of one or more microvessels (120). In other embodiments, a time period for testing drugs in the system is about 1 h to 24 h. In further embodiments, the system (100) described herein may be maintained for a period of time up to about 14 days.
  • Matrix Proteins Collagen (type I or type IV), gelatin, fibronectin and/or fibrin can be used to coat the arteriole and venular channels, with or without the aforementioned endothelial cell types.
  • collagen type I
  • fibrin In the microtissue compartment, both collagen (type I) and fibrin have been used to successfully support the development of in vitro microvessels networks (i.e. the channels) as they represent the dominant extracellular matrix protein in healing wounds and connective tissue.
  • Other natural and synthetic matrices could be employed, such as but not limited to, functionalized polyethylene glycol (PEG).
  • Materials to be used for the supportive structure/primary substrate layer for the microfabricated mold include, but are not limited to, glass and Si-wafers.
  • Photoresist sacrificial layer Materials used for the photoresist sacrificial layer are known to those of skill in the fabrication arts, whether they are positive or negative photoresist layers, or a combination thereof. These include, but are not limited to, SU-8.
  • Parylene Materials used for porous lower membrane to mimic the lymph drainage include, but are not limited to, Parylene.
  • parylene There are a number of derivatives and isomers of parylene including: Parylene N (hydrocarbon), Parylene C (one chlorine group per repeat unit), Parylene D (two chlorine groups per repeat unit), Parylene AF-4 (generic name, aliphatic flourination 4 atoms), Parylene SF (Kisco product), Parylene HT (AF-4, SCS product), Parylene A (one amine per repeat unit, Kisco product), Parylene AM (one methylene amine group per repeat unit, Kisco product), Parylene VT-4 (generic name, fluorine atoms on the aromatic ring), Parylene CF (VT-4, Kisco product), and Parylene X (a cross-linkable version, not commercially available).
  • Parylene N hydrocarbon
  • Parylene C one chlorine group per repeat unit
  • Parylene D two chlorine groups per repeat
  • the present invention also features methods and systems for introducing tissue (e.g., a microtissue) into prevascularized devices.
  • tissue e.g., a microtissue
  • a user may be able to add tissue to the sample after vascularization has occurred, which may allow for a user to quickly use a device, allow for development of automated loading systems, allow for growing larger organoids, etc.
  • a device may have an existing or developing vascular network, e.g., contained in hydrogel or other appropriate scaffold or support system.
  • the device features an empty tissue chapter that can be used as a media reservoir until used for tissue.
  • a user may introduce the tissue of interest to the device to be vascularized by the existing or developing vascular network.
  • a user may be able to introduce spheroids to the prevascularized device.
  • the present invention may feature a tissue receiving device (100) comprising one or more units.
  • Each unit may comprise a first microfabricated microfluidic channel (144) and a second microfabricated microfluidic channel (142) formed within a non-biological supportive material, and one or more microtissue compartments (105) formed within the non-biological supportive material.
  • at least one of the one or more microtissue compartments (105) comprises one or more living microvessels (120).
  • the one or more living microvessels (120) includes a first living microvessel that connects to the first microfabricated microfluidic channel (144) and the second microfabricated microfluidic channel (142), the first living microvessel having a lumen and running from the first microfabricated microfluidic channel (144), through at least one microtissue compartment (105), and to the second microfabricated microfluidic channel (142).
  • Cells e.g., endothelial cells, perivascular contractile perivascular cells, or a combination of endothelial cells and perivascular contractile perivascular cells
  • the biomimetic matrix may be combined with the biomimetic matrix and placed in at least one of the one or more microtissue compartments (105), such that the one or more living microvessels (120) are formed subsequent to placement of the cells combined with the biomimetic matrix within the at least one microtissue compartment (105).
  • the contractile perivascular cells (130) may wrap around at least a portion of each of the one or more living microvessels.
  • the one or more living microvessels (120) perfuse the cells combined with the biomimetic matrix and couple together the first microfabricated microfluidic channel (144) and the second microfabricated microfluidic channel (142).
  • the one or more living microvessels (120) allow for (i) delivery of nutrients from a fluid flowing from the first microfabricated microfluidic channel (144) to cells combined with the biomimetic matrix and (ii) removal of at least waste products from the fluid flowing to the second microfabricated microfluidic channel (142).
  • the fluid flows inside the lumen of the one or more living microvessels (120).
  • the one or more living microvessels (120) are selected from a group consisting of (i) one or more living lymphatic vessels and (ii) one or more living blood vessels.
  • the one or more living blood vessels are metabolically active.
  • the one or more living blood vessels may form a developing blood vessel network.
  • the one or more living blood vessels are capable of sprouting additional blood vessels.
  • the one or more living blood vessels may be stimulated to form in response to fluid flow rate in the plurality of microfabricated microfluidic channels (140).
  • the one or more microtissue compartments (105) are enclosed.
  • the one or more microtissue compartments (105) comprise one or more loading ports.
  • the one or more loading ports are configured to receive a tissue and are shaped accordingly.
  • the one or more loading ports are conical shaped.
  • the tissue may be a spheroid or an organoid and may be patient derived according to some embodiments.
  • the present invention may further feature a method of vascularizing a spheroid (or organoid e.g., a patient derived spheroid or organoid).
  • the method may comprise adding a first media to a tissue receiving device as described herein, and allowing the one or more living microvessels (120) to form over a period of time.
  • the first media is then aspirated out and a spheroid is added to at least one the one or more loading ports disposed on the one or more microtissue compartments (105).
  • a second media may then be added to the device.
  • the first media is within the first microfabricated microfluidic channel (144), the second microfabricated microfluidic channel (142), and the one or more microtissue compartments (105).
  • the second media comprises pro-angiogenic media.
  • the one or more living microvessels are allowed to form over a period of five to seven days, such that a vascular network is formed.
  • a vascular network is formed before the spheroid is added to at least one of the one or more loading ports (i.e., the spheroid is added to the device after one or more living microvessels are formed for 5-7 days).
  • the one or more living microvessels are allowed to form over a period of three to four days.
  • a vascular network is still forming when the spheroid is added to at least one of the one or more loading ports (i.e., the spheroid is added to the device after one or more living microvessels are formed for 3-4 days).
  • a vascular network comprises a plurality of interconnected living microvessels (120). The vascular network may sprout and penetrate the spheroid.
  • FIG. 17 shows a non-limiting example of a tissue receiving device described herein.
  • the device may comprise a central tissue chamber (e.g., central tissue chamber comprising one or more microtissue compartments (105)) with two adjacent and flanking media channels (i.e., a first microfabricated microfluidic channel (144) and a second microfabricated microfluidic channel (142)).
  • the central tissue chamber e.g., central tissue chamber comprising one or more microtissue compartments (105)
  • the central tissue chamber may comprise two or more or three or more loading ports.
  • the loading ports may comprise holes disposed through a top portion central tissue chamber of a microfluidic device described herein.
  • the one or more loading ports are disposed above the one or more microtissue compartments (105).
  • the one or more loading ports are configured to be within a well of a microplate.
  • the one or more loading ports may be filled with media to prevent the gel from drying out, keeping the tissue viable.
  • the media may be removed in order to transfer a tissue (e.g., a spheroid; e.g., an organoid) to the central tissue chamber.
  • a tissue e.g., a spheroid; e.g., an organoid
  • FIG. 18A shows a process of adding spheroids into existing mature vascular networks.
  • the process comprises lining microfluidic channels (140) (i.e., a first microfabricated microfluidic channel (144) and a second microfabricated microfluidic channel (142)) of a device described herein (i.e., a device comprising one or more loading ports), with endothelial cells (ECs) and normal human lung fibroblasts (LFs) to create a vascular network.
  • the microfluidic channels (140) may additionally be lined with contractile perivascular cells.
  • the vascular network may take about 7 days to form and be perfused.
  • the central tissue chamber e.g., central tissue chamber comprising one or more microtissue compartments (105)
  • the one or more loading ports are filled with the same media as the microfluidic channels.
  • the media may be aspirated out, and tissue (e.g., a spheroid) may be dropped into at least one of the one or more loading ports.
  • the central tissue chamber comprising the one or more loading ports may then be replenished with pro-angiogenic media to promote angiogenesis with the tissue (e.g., spheroid).
  • the vascular network will continue to sprout and penetrate the spheroid.
  • FIG. 18B shows a process of adding spheroids into a forming vascular network.
  • the process comprises lining microfluidic channels (140) (i.e., a first microfabricated microfluidic channel (144) and a second microfabricated microfluidic channel (142)) of a device described herein (i.e., a device comprising one or more loading ports), with endothelial cells (ECs) and normal human lung fibroblasts (LFs) to create a vascular network.
  • the microfluidic channels (140) may additionally be lined with contractile perivascular cells.
  • the vascular network takes about 7 days to form and be perfused.
  • the central tissue chamber of a microfluidic device and the one or more loading ports are filled with the same media as the microfluidic channels.
  • the tissue e.g., spheroid
  • the media is aspirated out, and the tissue (e.g., a spheroid) is dropped into at least one of the one or more loading ports.
  • the central tissue chamber comprising the one or more loading ports is replenished with pro-angiogenic media to promote angiogenesis with the tissue (e.g., spheroid).
  • the vascular network will continue to form and sprout towards the spheroid.
  • FIG. 19 shows an alternative embodiment of a tissue receiving device described herein.
  • the tissue receiving devices described herein may comprise at least two adjacent tissue chambers (e.g., the device may comprise two in vitro microphysiological devices (e.g., the Vascularized Micro-Organ (VMO) platforms or VMO-VA platforms) and a device central tissue chamber comprising the one or more loading ports).
  • the central tissue chamber comprising the one or more loading ports is sandwiched between the two in vitro microphysiological devices.
  • the one or more loading ports are configured to be within a well of a microplate.
  • the central tissue chamber comprising the one or more loading ports may be filled with media to keep the gel from drying out as well as to promote angiogenesis in the central tissue chamber.
  • FIG. 20 shows a process of vascularizing a central tissue chamber comprising one or more loading ports utilizing at least two VMO and/or VMO-VA platforms.
  • the process comprises lining microfluidic channels of two or more VMO and/or VMO-VA platforms with endothelial cells (ECs) and normal human lung fibroblasts (LFs) to create a vascular network.
  • ECs endothelial cells
  • LFs normal human lung fibroblasts
  • the outer media channels, the central tissue chamber and the platforms are filled with media.
  • the device as described in this embodiment, may be cultured until a vascular network forms in both of the platforms (i.e., in the microfluidic channels of the VMO and/or VMO-VA platforms described herein).
  • the in media in the central tissue chamber comprising one or more loading ports may be aspirated out and a tissue (e.g., a spheroid) may be dropped into at least one of the one or more loading ports.
  • a tissue e.g., a spheroid
  • Any tissue may be used in accordance with the present invention.
  • a coincident media well above the central tissue chamber is filled with pro-angiogenic media that will feed into the access ports. This will promote angiogenesis towards the central gel chamber.
  • parylene will be vapor deposited over a photoresist sacrificial layer followed by a photolithography step to etch the holes that represent the lymph channels (FIG. 1 and FIG. 2).
  • a negative resist SU-8 will be spin coated and patterned by photolithography to establish the primary channels representing the microfluidic vessel channels (i.e. arteriole and venule).
  • a second layer of SU-8 is then deposited to represent the arterio-venous communication ports that will sustain the capillary growth and fluid flow from arteriole to venule microfluidic channel via the microtissue in the microtissue compartment.
  • a capping layer of polydimethyl sioloxane (PDMS) is coated with a thin layer of SU-8 and bonded with the channel structures, which can correct for any unevenness which may occur from the multiple processing steps. Since PDMS is an elastomer and compliant it can generate an excellent seal as the thin layer of SU-8 is developed and exposed to UV light (similar to the process that has been previously presented).
  • the resulting device contains a series of channels/compartments grouped in threes (“arteriole” channel, microtissue compartment, and “venule” channel) in which all physical dimensions are design variables. Each channel/compartment can be connected to one or more reservoirs that serve as the source of fluid during the construction of the tissue and growth of the capillary network.
  • these reservoirs are illustrated as the ‘bulb’ like structures at one or both ends of the arteriole/venule channels and microtissue compartment.
  • the construction of the perfused microtissues can involve the following steps. A solution of collagen IV, collagen I, or appropriate matrix protein can be used to coat the arteriole, venule, and lymph vessels channels to mimic the basement membrane. A small volume of thrombin will then be added to a solution of fibrinogen, stromal cells, and/or endothelial cells, and immediately introduced into the microtissue matrix or microtissue compartment. If collagen or other biomimetic matrix is utilized, an alternate method of “gelling” or stiffening the matrix may be required.
  • the cellularized “tissue” then clots or polymerizes (5-10 minutes).
  • the presence of an air-liquid interface will create surface tension at the site of the ports and inhibit flow of the solution into the arteriole, venule, or lymph vessel channels.
  • the arteriole, venule, and lymph channels are seeded/coated with endothelial cells.
  • Arteriole and venule fluid flow will then be introduced, and the cellularized tissue will be allowed to develop and remodel, including endothelial cell migration and microvessel formation.
  • the microtissue compartment can be a closed environment, the only source of nutrients will come from the surrounding channels/vessels.
  • the microtissues for instance the stromal cells, create a metabolic deficit (hypoxic, acidic, secretion of angiogenic growth factors) and thus a pro-angiogenic environment that will induce microvessel growth from the arteriole and/or venule channels to meet metabolic demands.
  • Such induced microvessels will grow through the communication ports between the microtissue compartment and the arteriole and/or venule channels.
  • FIG. 1 shows one example of a fabrication scheme for in vitro metabolically active microtissues.
  • Device fabrication steps include: 1) spin and pattern a sacrificial photoresist (152) onto a glass slide (150); 2) deposit parylene (154) on sacrificial photoresist; 3) etch holes (156) in parylene which will serve as drains for lymph; 4) remove sacrificial photoresist to produce floating parylene membrane; 5) spin SU-8 onto glass and parylene and photopattern the microchannels (140) for passage of nutrients and flow between the microfluidic channels and through the microtissue; 6) cover top with layer of PDMS (158) to seal device.
  • the PDMS layer has holes for inlets and outlets.
  • FIG. 2A shows a cut-away view of microtissue channel highlighting the communication ports that will allow sprouting microvessels to penetrate tissue from either arteriole or venule channels, as well as the porous lower parylene layer (154) for lymph drainage.
  • FIG. 2B shows a top view highlighting channels and communication ports (160).
  • FIG. 2C shows venule (142) and arteriole (144) channels lined with collagen (I or IV) or fibronectin, and seeded with endothelial cells (135) which sprout through the channels — as microvessels (120) — to deliver nutrients to metabolizing tissue (contractile perivascular cells (135)) via the communication ports (160).
  • FIG. 3 shows another example of a scheme for the fabrication of an in vitro metabolically active microtissue.
  • the basic steps in the fabrication of the microfluidic platform include: Standard SU-8 photolithography process will be used to fabricate two micro-molds. First, a layer of SU-8 will be spin-coated onto a Si-wafer (RCA-1 cleaned and 2% HF treated). Then, a single mask photolithography step will pattern the tissue compartment. A similar SU-8 process will also be used to create molds for arteriole, lymph, and venule channels on another Si-wafer. The ports between the microtissue compartment and microfluidic channels will be created by spin-coating a second thick layer of SU-8 on the first layer.
  • the porous bottom layer of the lymph channels is also created by this layer of SU-8 with controlled diameter.
  • the second mask is then used to align and pattern high aspect ratio posts for creating ports and porous bottom layer.
  • a 3 mm thick layer of PDMS will be molded on the SU-8 mold to create the microtissue compartment.
  • a thin layer of PDMS will also be spun on the SU-8 molds for the arteriole, lymph, and venule channels and ports.
  • the PDMS microtissue compartment is then de-molded and bonded to the spun PDMS thin film by using 02 plasma.
  • the bonded multilayered PDMS device is then de-molded and holes punched to create an inlet/outlet to each channel (microtissue and microfluidic) before bonding to another 1 mm thick PDMS plate to seal channels.
  • the main difference in this design scheme compared to that in FIG. 1 is the placement of the microtissue compartment on a different plane (at a different height). This allows the endothelial cells to develop over a flat surface in the fluidic channels and avoids sharp curvatures that appear to limit cell attachment.
  • A-V arteriole-venule
  • A-V arteriole-venule
  • A-V arteriole-venule
  • each A-V system (including reservoirs 146) will span approximately 5 mm in the y-direction.
  • this platform could investigate the function of 2000 (mxn) similar or different types of microtissues.
  • m 10 microtissues along each A-V system in the z-direction, and 100 AV systems in the y-direction, or 1000 (mxn) microtissues on a single platform.
  • the venules are shown as 142, and arterioles as 144.
  • FIG. 7 includes a method by which long serpentine fluidic channels (140) can be strategically placed such that the pressure gradient is nearly constant across the individual microtissues (radial direction, z-axis), (7A, B) but significant along the microtissues (longitudinal direction, y-axis) (7B) or vice versa (7C).
  • the microfluidic device was fabricated by standard polydimethlsiloxane micro-molding.
  • the device consists of 2 fluid-filled microfluidic channels (arteriole and venule) on either side of a central metabolically active microtissue compartment consisting of normal human fibroblasts seeded (2x10 ® cells/ml) in a fibrin matrix.
  • a central metabolically active microtissue compartment consisting of normal human fibroblasts seeded (2x10 ® cells/ml) in a fibrin matrix.
  • human endothelial progenitor cell-derived endothelial cells (ECs) were used to line (1x10 6 cells/ml) the fluid-filled side channels (arteriole and venules) and allowed to migrate into and grow within the microtissue compartment via communicating ports (see FIG. 2).
  • ECs were randomly distributed throughout the microtissue compartment with the fibroblasts.
  • a pressure gradient (2 mm H 2 0) across the tissue compartment was applied once obvious network formation was identified.
  • Flow in formed microvessels was assessed by adding 1 pm diameter polystyrene fluorescent beads into the microfluidic channels and tracking their movement in the formed microvessels across the tissue compartment.
  • Multiphoton microscopy was used to image devices stained for CD31 markers (EC marker) and DAPI (nuclei stain).
  • the three microfabricated compartments can be formed so that all three are on the same horizontal level.
  • the arteriole and venule channels can be formed so that they are both at different levels to the tissue compartment, for instance, these channels are formed above the level of the microtissue compartment.
  • the arteriole and venule channels are not only at different levels from the microtissue compartment but also on different levels from each other.
  • the arteriole and venule channels do not have to run parallel throughout their entire lengths to the microtissue compartment.
  • FIG. 6 shows a view of such a fabricated 3D cell system as viewed under a microscope. Here, only a small region of the venule channel (142) and arteriole channel (144) is shown running parallel to the microtissue compartment (105). Also shown are the reservoirs (146) for the microtissue compartment (reservoirs for the venules and arterioles are not depicted on this figure).
  • the microtissue compartment can be alternate shapes (e.g., diamonds or tear-drops) rather than one long central microtissue channel/compartment (FIG. 8). This design facilitates the separation of the tissues by diffusion, although the microvessels may (but are not required to) still penetrate between the microtissue compartments for a fluidic connection.
  • FIG. 9A-9B Single units (FIG. 9A) or multiple units (FIG. 9B) are arrayed, with each unit containing 3 linked tissue compartments (in the x-direction) that connect two microfluidic lines in the y-direction.
  • the upper channel represents the high pressure “arterial” side of the circulation, while the lower channel represents the low pressure “venular” side (FIGs. 9C-9D).
  • the tissue compartment is loaded with ECM, and fibrin to control gelation, along with endothelial cells (EC) (lentivirally transduced to express fluorescent proteins) and stromal cells, including fibroblasts, and pericytes (generated in the lab and confirmed for NG2 expression).
  • EC endothelial cells
  • stromal cells including fibroblasts, and pericytes (generated in the lab and confirmed for NG2 expression).
  • Endothelial Colony-Forming Cell-derived EC (ECFC-EC) was routinely used, although other EC types, including HUVEC and iPSC-derived EC, work equally well.
  • a capillary bed forms in the compartment over 4-5 days, anastomosing with EC lining the arteriole and venule to form a complete, EC-lined vascular network (FIGs. 9E-9G). Vessels generally have an internal diameter of 20-50 pm, which is consistent with small arterioles.
  • a hydrostatic head of pressure (FIG. 9A, V1-V4) then drives medium through the channels, through the capillary bed - supplying surrounding cells with nutrients - and then out of the low-pressure side.
  • the hydrostatic head regulates the flow rate, which is set at a physiological rate for capillaries of -0.33 mm/s.
  • the higher throughput version (FIG. 9B) works similarly.
  • 70 kDa FITC-labeled dextran is routinely flown through the vessels to confirm their patency (FIG. 9F).
  • Stromal cells help to generate a suitable ECM and pericytes wrap around the microvessels as they do in vivo and contribute to a collagen IV-rich basement membrane (FIG. 9H-9I). These vessels have a permeability coefficient of 1.2 x 10 ® cm/s, which is similar to what is seen for capillaries in vivo (FIG. 9J).
  • vasoconstrictors L-NMMA (nitric oxide synthase inhibitor) and phenylephrine (a1- adrenoreceptor agonist), were administered across a six-dose curve up to the reported peak plasma concentration of each (700 nM and 8.8 mM, respectively).
  • the optimal time point for measuring vasoactivity was determined to be 4 hours post drug addition.
  • nifidipine Ca 2+ channel inhibitor
  • nitroprusside Natric Oxide donor
  • Perivascular cells tested for the VMO-VA platform It was originally proposed to incorporate commercial lines of perivascular cells (SMC) into the vascularized micro-organ (VMO) platform and leverage the self-assembly process to create a vasoactive network.
  • SMC perivascular cells
  • Two primary SMC lines purchased from Lonza were tested first: Human Umbilical Artery SMC (“UA-SMC”) and Human Aortic SMC ( “AO-SMC”). To track the morphology and vessel wrapping capacity of these cells in the VMO platform, both SMC lines were expanded and transduced lentivirus expressing fluorescent tags (FIG. 16; bottom two panels).
  • Endothelial cells EC
  • stromal cells normal human lung fibroblasts, “LF”
  • EC Endothelial cells
  • stromal cells normal human lung fibroblasts, “LF”
  • EC Endothelial cells
  • LF normal human lung fibroblasts
  • the figures are representative only and the claims are not limited by the dimensions of the figures
  • the reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.
  • descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of or “consisting of is met.

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Abstract

La présente invention concerne une plateforme de réseau vasoactif (VMO-VA). La plate-forme tient dans la paume de la main et contient des tissus individuels, chacun doté d'un réseau vasculaire humain fictif fu I ly-fu. Les microvaisseaux composant le réseau sont enveloppés par des cellules périvasculaires contractiles et sont perfusés avec du sang ou un substitut sanguin. Les cellules périvasculaires contractiles répondent aux médicaments vasodilatateurs et vasoconstricteurs de la même manière que les vaisseaux sanguins dans le corps. Cette plateforme peut être utilisée pour évaluer les effets vasoactifs souhaitables ou indésirables des médicaments sur les vaisseaux sanguins, et possède donc des applications pour caractériser à la fois l'efficacité (par exemple, la dilatation des vaisseaux sanguins dans l'hypertension) et la sécurité dans le cadre du développement de médicaments.
PCT/US2022/038551 2021-07-27 2022-07-27 Système microphysiologique in vitro de système vasculaire vasoactif Ceased WO2023009645A1 (fr)

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CN116045878A (zh) * 2023-03-07 2023-05-02 中国人民解放军军事科学院军事医学研究院 一种血管管径检测设备

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