[go: up one dir, main page]

WO2025122436A1 - Organe sur puce multicouche dynamique (dynamoc) pour la simulation complète de la physiopathologie des organes - Google Patents

Organe sur puce multicouche dynamique (dynamoc) pour la simulation complète de la physiopathologie des organes Download PDF

Info

Publication number
WO2025122436A1
WO2025122436A1 PCT/US2024/058124 US2024058124W WO2025122436A1 WO 2025122436 A1 WO2025122436 A1 WO 2025122436A1 US 2024058124 W US2024058124 W US 2024058124W WO 2025122436 A1 WO2025122436 A1 WO 2025122436A1
Authority
WO
WIPO (PCT)
Prior art keywords
chamber
biological
flow rate
flow
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/058124
Other languages
English (en)
Inventor
Ishita TANDON
Kartik Balachandran
Cody GISH
Hemali GAURI
Amanda Walls
Alexis APPLEQUIST
Lais Andrade FERREIRA
Gustavo Vaca DIEZ
Connor ROBINSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Arkansas at Fayetteville
University of Arkansas at Little Rock
Original Assignee
University of Arkansas at Fayetteville
University of Arkansas at Little Rock
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Arkansas at Fayetteville, University of Arkansas at Little Rock filed Critical University of Arkansas at Fayetteville
Publication of WO2025122436A1 publication Critical patent/WO2025122436A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N5/0062—General methods for three-dimensional culture
    • 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
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • 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
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture

Definitions

  • the present disclosure pertains to a microfluidic system.
  • the microfluidic system includes: (1) a first chamber with an inlet and an outlet, where the first chamber is operable to flow a biological fluid through the inlet and the outlet at a first flow rate; (2) a first porous membrane in fluid communication with the first chamber; (3) a second chamber in fluid communication with the first porous membrane, where the second chamber is operable to serve as a reservoir for one or more biological materials; (4) a second porous membrane in fluid communication with the second chamber; and (5) a third chamber in fluid communication with the second porous membrane, where the third chamber includes an inlet and an outlet, and where the third chamber is operable to flow a biological fluid through the inlet and the outlet at a second flow rate.
  • Additional embodiments of the present disclosure pertain to methods of forming a biological component in a microfluidic system of the present disclosure.
  • such methods include: (1) flowing a biological fluid through the inlet and the outlet of the first chamber at a first flow rate; (2) placing one or more biological materials in the second chamber, and (3) flowing a biological fluid through the inlet and the outlet of the third chamber at a second flow rate.
  • FIGS. 1A-1C provide illustrations of a microfluidic system in accordance with various embodiments of the present disclosure.
  • FIG. 2 is a schematic of a cross-section of an aortic valve along with the mechanical forces (top left), cross-section of the proposed chip structure and assembly (top right), and the assembly of the whole valve-on-chip with the flow loops.
  • FIGS. 3A-3C provide schematics of a prototype of a dynamic multilayered organ-on- chip (DynaMOC) with separate chambers and assembled chip without the cellular components.
  • DynaMOC dynamic multilayered organ-on- chip
  • FIG. 4 is a schematic of a prototype of DynaMOC with molds and PDMS Chip Pieces.
  • FIG. 6A is a wall shear stress profile of an oscillatory flow showing 10 dynes/cm 2 shear stress at a porous membrane.
  • FIG. 6B is a shear stress and volumetric flow rate at a point in the center of a porous membrane over time for oscillatory flow.
  • FIG. 6C is a wall shear stress profile of the laminar flow showing 20 dynes/cm 2 shear stress at the porous membrane.
  • FIG. 7 shows strain experienced by the porous membrane as a function of programmed stretch on a custom-built stretcher device to achieve 10% cyclic strains for the porous membranes.
  • FIG. 8 shows a schematic representing the alternative design for a high throughput version of the Chip.
  • FIG. 9 shows a schematic representing the alternative designs for modifying the middle chamber.
  • FIG. 10 is a schematic where DynaMOC serves as a cross-section of a blood-brain barrier.
  • FIGS. 11A-11C show different variations of first and third chambers of the microfluidic systems of the present disclosure, where the inlet and the outlet are in lateral positions (FIGS. 11A-11B), and where the inlet and the outlet are in angled positions (FIG. 11C).
  • FIG. 12 shows additional variations of the microfluidic systems of the present disclosure.
  • heart valve diseases are on the rise with lifestyle changes and an increase in the aging population in the world. With no treatment alternatives to surgical intervention for treating severe valve diseases, organs on chip (OOCs) can help in the development of early diagnosis and treatment strategies.
  • OOCs organs on chip
  • microfluidic system 10 generally includes a first chamber 12, a first porous membrane 16, a second chamber 18, a second porous membrane 20, and a third chamber 22.
  • First chamber 12 generally includes an inlet 13 and an outlet 14. Additionally, first chamber 12 is operable to flow a biological fluid through inlet 13 and outlet 14 at a first flow rate.
  • First porous membrane 16 is in fluid communication with the first chamber 12 and second chamber 18. In some embodiments, first porous membrane 16 is between the first chamber 12 and the second chamber 18. In some embodiments, first porous membrane 16 also includes a plurality of apertures 17 to facilitate fluid flow from first chamber 12 to second chamber 18.
  • Second chamber 18 is in fluid communication with the first porous membrane 16.
  • second chamber 18 is generally operable to serve as a reservoir for one or more biological materials.
  • second chamber 18 includes a plurality of compartments.
  • second chamber 18 includes at least two compartments 18A and 18B.
  • each compartment is operable to serve as a reservoir for one or more biological materials.
  • second chamber 18 is positioned between the first porous membrane 16 and the second porous membrane 20.
  • Second porous membrane 20 is in fluid communication with the second chamber 18 and the third chamber 22.
  • second porous membrane 20 includes a plurality of apertures 21 to facilitate fluid flow from second chamber 18 to third chamber 22.
  • second porous membrane 20 is between the third chamber 22 and the second chamber 18.
  • Third chamber 22 is in fluid communication with second porous membrane 20.
  • Third chamber 22 generally includes an inlet 23 and an outlet 24. Additionally, third chamber 22 is operable to flow a biological fluid through inlet 23 and outlet 24 at a second flow rate.
  • inlet 13 and outlet 14 of first chamber 12 are in lateral positions. In some embodiments, inlet 13 and outlet 14 of first chamber 12 are in angled positions. In some embodiments, inlet 23 and outlet 24 of first chamber 22 are in lateral positions. In some embodiments, inlet 23 and outlet 24 of first chamber 22 are in angled positions.
  • Additional embodiments of the present disclosure pertain to methods of forming a biological component by utilizing the microfluidic systems of the present disclosure.
  • the methods of the present disclosure include flowing a biological fluid through the inlet 13 and outlet 14 of the first chamber 12 at a first flow rate; placing one or more biological materials in second chamber 18; and flowing a biological fluid through inlet 23 and outlet 24 of the third chamber 22 at a second flow rate.
  • such processes result in the formation of a biological component in the microfluidic system.
  • First chamber 12 and third chamber 22 may be operable to flow various biological fluids through their respective inlets and outlets. Additionally, the methods of the present disclosure may be utilized to flow various biological fluids through first chamber 12 and third chamber 22.
  • the biological fluids include, without limitation, cell culture fluids, cells, cellular matrices, or combinations thereof.
  • Second chamber 18 may be operable to serve as a reservoir for various biological materials. Additionally, the methods of the present disclosure may place various biological materials in second chamber 18.
  • the biological materials include, without limitation, hydrogels, cells, collagen, extracellular matrices, glycosaminoglycans, elastin, laminin, fibronectin, organoids, or combinations thereof.
  • the biological materials of the present disclosure can include combinations of different extracellular matrix components along with cells.
  • the biological materials of the present disclosure can include valve interstitial cells in a matrix made with collagen, glycosaminoglycans, and elastin.
  • the biological materials of the present disclosure can also include organoids.
  • First chamber 12 and third chamber 22 may be operable to flow biological fluids at various flow rates. Additionally, the methods of the present disclosure may flow biological fluids through first chamber 12 and third chamber 22 at various flow rates.
  • the first flow rate of the first chamber 12 and the second flow rate of the third chamber 22 represent the same flow rates. In some embodiments, the first flow rate of the first chamber 12 and the second flow rate of the third chamber 22 represent different flow rates. In some embodiments, one of the first or second flow rates includes an oscillatory flow rate. In some embodiments, the other of the first or second flow rate includes a steady flow rate. In some embodiments, at least one of the first or second flow rates mimics fluid shear stress.
  • First and second flow rates may be actuated in first and third chambers in various manners.
  • first and second flow rates may be actuated by one or more pumps.
  • the pumps include, without limitation, peristaltic pumps, syringe pumps, or combinations thereof.
  • the microfluidic systems of the present disclosure can also include one or more biological sensors that are operable to sense one or more conditions.
  • the methods of the present disclosure also include a step of sensing one or more conditions through one or more biological sensors.
  • the biological sensors of the present disclosure may be utilized to sense various conditions.
  • the conditions include, without limitation, pH, oxygen levels, lactic acid levels, glucose levels, electrical resistance, or combinations thereof.
  • the microfluidic systems of the present disclosure may be operable to form a biological component within the system. Additionally, the methods of the present disclosure may be utilized to form various biological components in a microfluidic system.
  • the biological component is an organ, an organ component, a tissue, or combinations thereof.
  • the biological component is a heart tissue, heart valve, placenta, a gastrointestinal tissue, a liver, a kidney, a lymphatic system, a blood-brain barrier, or combinations thereof.
  • the microfluidic system is operable to mimic the tissue structure, cellular components, and mechanical environment of the biological component.
  • the biological component can simulate organs and tissues that have differential flow requirements on either side of that tissue, such as seen in the kidney, liver or in the lymphatic system.
  • the biological component is a heart tissue.
  • the heart tissue mimics heart blood flow, contraction-relaxation of a beating heart, or combinations thereof.
  • the biological component is a heart valve.
  • the biological fluids of the first chamber 12 and the third chamber 22 include valve endothelial cells (VEC).
  • the biological materials of the second chamber 18 include, without limitation, valve interstitial cells (VICs), collagen, glycosaminoglycans, or combinations thereof.
  • the first flow rate of the first chamber 12 includes an oscillatory flow rate to simulate an aortic side of an aortic valve.
  • the second flow rate of the third chamber 22 includes a steady flow rate to simulate a ventricular side of an aortic valve.
  • the biological fluids of the first chamber 12 can include peripheral blood mononuclear cells while first porous membrane 16 is seeded with vascular endothelial cells and/or valvular endothelial cells.
  • second chamber 18 can include vascular smooth muscle cells and/or valve interstitial cells (VICs) to simulate the artery and/or valve.
  • VIPs valve interstitial cells
  • such a biological component can be used to specifically assess the monocyte and lymphocyte interaction, attachment, and infiltration in various diseases, such as atherosclerosis, valve calcification, or combinations thereof.
  • the biological component is a liver.
  • the first flow rate of the first chamber 12 is operable to simulate blood flow.
  • the biological fluids of the first chamber 12 include sinusoidal endothelial cells and Kupffer cells to mimic a vascular layer.
  • the biological materials of the second chamber 18 include, without limitation, stellate cells, hepatocytes, collagen, fibronectin, organoids, glycosaminoglycan, laminin, or combinations thereof.
  • the biological fluids of the third chamber 22 include hepatic cells, biliary endothelial cells, and cholangiocytes to mimic a bile duct.
  • the second flow rate of the third chamber 22 is operable to simulate bile duct flow.
  • the biological component is a blood-brain barrier.
  • the third chamber 22 can serve as a vascular channel that includes brain microvascular endothelial cells along with fluid flow.
  • at least half of the second chamber 18 can serve as the neural chamber that includes a sequential assembly of pericytes, astrocytes, microglia, and neurons in an extracellular matrix to simulate the complete blood-brain barrier.
  • at least another half of the second chamber 18 can serve as a lid or cover for the culture, or as a media reservoir for the cells.
  • the components of the microfluidic systems of the present disclosure may include various compositions.
  • the microfluidic system components include one or more elastic polymers.
  • the elastic polymers include polydimethylsiloxane (PDMS).
  • microfluidic systems and methods of the present disclosure have numerous advantages. For instance, in some embodiments, the microfluidic systems and methods of the present disclosure can be utilized to effectively emulate a human organ or tissue on a chip.
  • the microfluidic systems and methods of the present disclosure can have numerous applications.
  • the microfluidic systems and methods of the present disclosure can be utilized to evaluate disease mechanisms, disease progression, treatment strategies, immune responses, infectious and degenerative diseases, comorbidities, drug efficacy, drug toxicity, or combinations thereof.
  • the microfluidic systems and methods of the present disclosure can be utilized to evaluate personalized medicine treatments.
  • the microfluidic systems and methods of the present disclosure can be utilized to evaluate long-term longitudinal imaging and assessment of cells.
  • the microfluidic systems and methods of the present disclosure can be utilized to evaluate drug efficacy and/or drug toxicity.
  • several chemicals and/or small molecules may be introduced to the biological components in microfluidic systems (e.g., by fluid flow through first chamber 12 and/or third chamber 22) to assess their effects on the biological components (e.g., genetic toxicity of cancer drugs and/or disease causing risk of certain metabolites).
  • Example 1 Development of a dynamic multi-layered organ-on-chip (DYNAMOC)
  • OOC organ-on-chip
  • 3D three-dimensional
  • the design goal in this Example included mimicking the structure and function of complex multilayered organs, such as the heart valve, placenta, and the gut.
  • heart valve diseases are on the rise with lifestyle changes and an increase in the aging population in the world.
  • OOCs can help in the development of early diagnosis and treatment strategies. It is, therefore, imperative to understand key disease mechanisms and processes involved in early disease progression, which requires the development of benchtop models that closely mimic the structure and function of the heart valve.
  • FIG. 2 the complex tissue structure, cellular components, and mechanical environment of the heart valve
  • DynaMOC dynamic multilayered organ on chip
  • DynaMOC is a multi-layered cell culture platform, including 3 separate chambers stacked onto each other (FIGS. 3A-3C).
  • the top and bottom chambers are designed to facilitate fluid flow and cell culture and a middle chamber that can facilitate the incorporation of hydrogel and cells.
  • the three chambers are separated by thin microporous membranes which are fabricated in-house and can facilitate separate analysis of all cell types.
  • the three chambers in the DynaMOC can facilitate the culture of three separate cell types with their respective matrices.
  • the two flow chambers are attached to pumps to facilitate similar or different fluid flow patterns and fluid shear stresses.
  • the fluid shear can be a mimic of the blood flow in the heart while the strain application can mimic the contraction-relaxation of a beating heart.
  • the fluid shear can also mimic the blood flow and nutrient flow in the intestinal tissue while the stretch can mimic the peristaltic movement of the gut lining for a guton-chip system.
  • the top and bottom flow channels can simulate different fluid flow profiles.
  • the chip is designed to be able to facilitate physiological levels of flow rate (simulating blood flow through the aortic valve).
  • DynaMOC can undergo stretching to incorporate mechanical strains. It is designed to experience physiological levels of cyclic strains (simulating up to 30% of strain experienced by the aortic valve).
  • the entire chip is made from an elastic polymer named poly dimethylsiloxane (PDMS).
  • PDMS is oxygen permeable to facilitate long-term cell culture, self-adhering such that uncured PDMS can be used to “glue” two PDMS-based components together, and elastic so it can facilitate the application of cyclic strains.
  • the middle chamber is designed in two parts that fit onto each other and can be attached together. The top chamber is attached to a middle chamber and bottom chamber is attached to a separate middle chamber slightly bigger in size. The two middle chambers fit onto each other and can be shut closed using plasma activation and curing PDMS.
  • DynaMOC can serve as an aortic valve on chip (VOC) (FIG. 2).
  • VOC aortic valve on chip
  • the top and bottom flow chambers can be seeded with side-specific valve endothelial cells.
  • the top flow chamber can be attached to a syringe pump to facilitate 10 dynes/cm 2 oscillatory shear stress on endothelial cells and simulate the aortic side of the aortic valve.
  • the bottom flow chamber can be attached with a peristaltic unidirectional flow system to facilitate 20 dynes/cm 2 pulsatile shear stress on endothelial cells to simulate the ventricular side of the aortic valve.
  • the middle chamber can be seeded with valve interstitial cells in a bilayered matrix comprising of collagen and glycosaminoglycans to simulate fibrosa and spongiosa layers of the aortic valve.
  • the chip can then be sealed shut and mounted onto a cyclic stretcher device to simulate physiological 10% cyclic strains.
  • the fluid shear stresses, cyclic strains, different cell types, and cell-matrix interaction are all key factors that affect the structure, function, and disease processes of the aortic valve cells.
  • DynaMOC is one of a kind and can fill the gap of understanding how all these key factors interact to progress the disease and elucidate important players that can be targeted to develop early treatment strategies.
  • Several chemical and small molecules can also be introduced to the cells using the fluid flow to assess their effect on cells, for example, genetic toxicity of cancer drugs and disease causing risk of certain metabolites.
  • DynaMOC can be used to assess several existing drugs and treatment strategies on patient-derived cells to further personalized medicines.
  • DynaMOC is a multi-chamber PDMS based device capable of 2 separate fluid flow profiles, cyclic strains, cell co-cultures, and incorporation of matrix.
  • FIGS. 3A-3C and 4 illustrate the construction and assembly of DynaMOC.
  • the top and bottom flow chambers are identical in structure and dimensions.
  • the flow chamber is 7.8 mm x 50 mm with outer walls being 1.5 mm each.
  • the middle chamber has two parts (i.e., inner and outer middle chambers).
  • the inner middle chamber is 7 mm x 22 mm in size and includes only “walls” with a height of 2 mm and thickness of 1 mm.
  • the outer middle chamber is 8.5 mm by 23.5 mm in size and includes 1 mm walls that are 2.2 mm in height, allowing a snug fit and closure.
  • 3D molds can be made using 3D printing or CNC router.
  • the molds can be reused to fill uncured PDMS in the shapes of top and middle chambers. Briefly, a 1 : 10 mixture of curing agent to the base of the Sylgard 184 is mixed and degassed before filling the molds. The molds are kept in a vacuum for 20 mins before curing at a 37 °C oven overnight. The PDMS pieces are then de-molded, and the molds are cleaned with 50% ethanol for reuse.
  • 2 PDMS-based 10 pm thick, 5 mm x 20 mm porous membranes are fabricated in-house using photolithography and soft lithography techniques. Briefly, a mask is constructed for a 5 mm x 20 mm design containing pores that are 5 pm in diameter and are spaced 50 pm apart from center to center. A silicon wafer is then etched using that mask and an aligner. The silicon wafer is spun-coated with photoresist and PDMS. Porous membranes are then attached to 5.4mm x 3.6 mm silicon membranes using PDMS. Silicon membranes used for this are pre-cut to size using a Silver Bullet Cutter device. The photoresist layer is dissolved using acetone to reveal a 10 pm thick porous membrane. Silicon wafers are washed using acetone and along with ethanol and methanol for reuse.
  • the inner middle chamber and top flow chamber are attached to the opposite sides of one porous membrane.
  • a very thin layer of uncured PDMS is used as glue to attach different PDMS- based pieces together and cured in a 37°C oven overnight to facilitate attachment.
  • the outer middle chamber and bottom flow chamber are attached on opposite sides of the other porous membrane. The constructs can then be sterilized using ethylene oxide.
  • Example 1,2 Addition of co-culture and multilayered hydrogel
  • VECs valve endothelial cells
  • FIG. 2 the inner middle chamber
  • the outer middle chamber can be used to completely sandwich the gel between the top and bottom chambers using uncured PDMS. Once the PDMS is cured, the entire chip is assembled and ready for the application of stretch and flow.
  • the multilayered hydrogel is assembled layer by layer to mimic the fibrosa and spongiosa layers of the aortic valve (FIG. 2).
  • Collagen 1 is used to construct the fibrosa mimicking layer.
  • Collagen 1 mixed with glycosaminoglycans (GAG) Hydrouronic acid and Chondroitin sulfate
  • GAG glycosaminoglycans
  • VICs valve interstitial cells
  • [0080] Defined concentrations of Collagen 1 , Hyaluronic acid, and Chondroitin sulfate are used to mimic healthy and diseased compositions of the aortic valve layers.
  • the hydrogel is attached to the PDMS using benzophenone, polydopamine or Cell-Tak.
  • DynaMOC can be used to study valve disease progression. Current plans to use DynaMOC include constructing a healthy and diseased version of the aortic valve and exploring the different mechanisms contributing to and involved in initiating the disease process in the valves.
  • the healthy chip can consist of VECs and VICs derived from healthy human valves.
  • the chip can also have optimized amounts of collagen 1 and GAGs that mimic the matrix makeup of a healthy valve. Physiological levels of shear stress and cyclic strains can be applied to the chip.
  • patients with aortic stenosis can be used as the cell source.
  • the diseased composition of the matrix can be used to mimic the collagen degradation and GAG enrichment observed during aortic stenosis. Pathological shear stresses and cyclic strains can be applied to the chip. Healthy and diseased media compositions can be used on the healthy and diseased chips, respectively, to provide appropriate chemical cues, nutrition for cells, and flow shear stresses.
  • the chips can be cultured in these healthy and diseased conditions over time and characterized for diseased progression using various assays. For phenotypic and functional characterization of cells based on their protein makeup, cells from the two endothelial layers and the hydrogel layer can be isolated for assays like flow cytometry or lysed to collect proteins for western blots. Media flowed through the chip can be collected for western blots and ELISA for assessing the secreted factors from the cells. Chips can also be used to carry out MTS assay to assess cell metabolism and viability. VOCs can be stained or immunolabeled for visual assessment of cell phenotype and function. The hydrogel portion can also be removed, embedded in OCT, and sectioned for histological examination.
  • DynaMOC can also be used to simulate other organ types, for example, kidney, and assess multi-organ diseases and comorbidities with aortic valve disease.
  • renal tubular epithelial cells can be seeded onto the top chamber, endothelial cells seeded onto the bottom chamber, and connected in series with the pulsatile shear fluid flow patterns of the VOC.
  • the middle chamber on the kidney on chip is seeded with collagen, laminin, and fibronectin-based matrix. This setup with valve and kidney on chip attached in series can be used to assess the mechanism of increased aortic valve disease associated with renal failure.
  • the middle chamber can be shortened for the matrix and the bottom flow chamber can be the vascular channel with endothelial and immune cells.
  • This chip can also be used as a placenta on chip with the top and bottom flow chambers simulating maternal and fetal blood. Synctiotrophoblasts/cytotrophoblasts (or the placental barrier cells) can be seeded on the maternal flow side and any fetal cells. For example, human vascular endothelial cells and cardiomyocytcs can be seeded on the fetal side.
  • the middle chamber can house the collagen matrix and connective tissue.
  • Liver on chip can be constructed by using one flow chamber as the vascular layer (simulating blood flow) and the other flow chamber as the hepatic layer (simulating bile duct).
  • Sinusoidal endothelial cells can be seeded on the vascular side of the porous membrane along with Kupffer cells.
  • Stellate cells and Hepatocytes can be seeded in the middle chamber along with the matrix made up of collagen, fibronectin, glycosaminoglycan and laminin.
  • Biliary endothelial cells and cholangiocytes can be seeded on the hepatic side of the porous membrane.
  • DynaMOC can function as any OOC that requires the use of two separate flow types, mechanical strains, cellular co-cultures, and cells embedded in a matrix.
  • DynaMOC can function as a blood-brain barrier with the third chamber (bottom flow channel) serving as a vascular channel and half of the second chamber (middle chamber) serving as the neural chamber.
  • the vascular chamber will consist of brain microvascular endothelial cells along with fluid flow.
  • the neural chamber can consist of a sequential assembly of pericytes, astrocytes, microglia, and the neurons in an appropriate extracellular matrix to simulate the complete blood-brain barrier.
  • the other half can serve as a lid/cover for the culture or as a media reservoir for the cells.
  • DynaMOC The versatility of DynaMOC and its easy construction and assembly allows the chip to be able to study diseases pertaining to various organs it can simulate. For example, by altering the matrix composition of the liver-on-chip and adding immune cells in the vascular chamber, a disease model for liver fibrosis can be constructed.
  • This chip is a multilayered multiplex dynamic platform for a comprehensive simulation of organ pathophysiology.
  • a novelty of this invention lies in the ability of the platform to be able to include hydrogel assembly, co-culture, two types of flow, and strain application simultaneously in a single platform. All these components, which are pivotal to the organ microenvironment, have never been included in a single organ-on chip design.
  • this is a versatile chip that can serve not just as a VOC but also as any organ on chip as it can mimic the complex tissue architecture of most organs and their disease processes.
  • the easy incorporation of mechanical and chemical cues also allows it to study multiple organ pathophysiologies and test drugs for those diseases.
  • the design flexibility of this chip enables the incorporation of several sensors for real-time monitoring and live imaging for facile assessments.
  • Organs that utilize two separate flow profiles e.g., blood and urine for the kidney; blood and food passage for the gut; maternal blood and fetal blood for the placenta, etc.
  • mechanical stretch e.g., the beating of the heart, peristaltic movements of the gut lining, contractions of the placenta, etc.
  • various cell types and a matrix can be simulated using the DynaMOC chip.
  • DynaMOC can facilitate physiological and pathological mechanical cues to study the healthy and diseased functioning of various organs. Different layers can be separated to assess how the various components of the organ are impacted during health and disease.
  • this chip can be equipped with various sensors for monitoring various physiological parameters (e.g., pH, O2 permeation, lactic acid and glucose production and consumption, transendothelial electrical resistance, etc.).
  • physiological parameters e.g., pH, O2 permeation, lactic acid and glucose production and consumption, transendothelial electrical resistance, etc.
  • the incorporation of a bioelectrochemical sensing module can be facilitated with this chip.
  • DynaMOC facilitate live imaging of the cells seeded on the flow channels. Future validations and size changes can ensure that all the layers can be monitored longitudinally via live imaging for easy assessment. Changes in size can also be made to accommodate more severe flow rates and mechanical strains. Current dimensions are optimized for pathological fluid flow and loading of the aortic valve. PDMS itself can be mixed in various ratios to construct a chip with higher elasticity and facilitate higher strain loading.
  • the chip molds can be printed in parallel side by side to facilitate a high throughput assembly of the chips such that a single process of chip production can generate 3 or more chips at a time (FIG. 8).
  • the middle chamber itself can be further divided into multiple middle chambers, either horizontally or vertically, to assemble more complex extracellular matrices (FIG. 9).
  • the inner middle chamber can be constructed in sections by attaching a porous membrane after each section.
  • the outer middle chamber can remain the same and enclose the inner middle chamber.
  • aortic valve tissue gets calcified with age.
  • the calcified and noncalcified regions of the valve present different phenotypes and matrix makeups.
  • a chip can then be constructed with the middle chamber separated with a vertical porous membrane and calcified versus noncalcified regions can be constructed separately for further assessment.
  • the inlet and outlet on the flow chambers can be placed laterally.
  • This modification paired with L shaped elbow fittings, allows access to both the top and bottom channel without the need for manual handling of the entire system.
  • this configuration can allow for media changes and sample collection from the fluid channel without the need to invert the chip to access the flow channel on the other side.
  • the inlet and outlet ports can also be placed on an angle to accommodate specific and unique flow needs.
  • the angle of the elbow fittings can be changed and oriented as per convenience of the user, making the system more user-friendly.
  • the elbow fitting orientation can be changed as per space availability as well.
  • Organ-on-chip is a 3-dimensional (3D) system that offers the scope to incorporate cells in a relevant substrate, is conducive to appropriate biophysical and biochemical stimuli, and is sustainable and reproducible.
  • This Example pertains to mimicking the structure and function of various organs and tissues, such as the heart valve.
  • Heart valve diseases are on the rise with lifestyle changes and an increase in the aging population in the world.
  • OOCs that are able to mimic the complex tissue structure, cellular components, and mechanical environment of complex multilayered, dynamic tissues.
  • Examples of such tissues include the heart valve, kidney, liver and placenta, among others.
  • DynaMOC fills a research gap by being capable of incorporating the different cellular and acellular components, different structural complexities, and mechanical forces that make up the intricate and dynamic environment of the heart valves.
  • the OOC Applicant developed is a multilayered multiplex dynamic platform for a comprehensive simulation of organ pathophysiology.
  • the fluid shear stresses, cyclic strains, different cell types, and cell-matrix interactions are all key factors that affect the structure, function, and disease processes of the aortic valve cells.
  • This OOC is one of a kind and can fill the gap of understanding how all these key factors interact to progress the disease and elucidate important players that can be targeted to develop early treatment strategies. Given the complex multilayered structure of DynaMOC, it can also be used to simulate other organ types, such as the kidney, liver, placenta, and/or gut.
  • DynaMOC can function as any organ-on-chip that requires the use of two separate flow types, mechanical strains, cellular co-cultures, and cells embedded in a matrix.
  • the versatility of the chip and its easy construction and assembly allows the chip to be able to study diseases pertaining to various organs it can simulate.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physiology (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un système microfluidique incluant : une première chambre avec une admission et une évacuation, la première chambre étant fonctionnelle pour faire circuler un fluide biologique à travers l'admission et l'évacuation à un premier débit; une première membrane poreuse en communication fluidique avec la première chambre; une deuxième chambre en communication fluidique avec la première membrane poreuse, la deuxième chambre étant fonctionnelle pour servir de réservoir pour une ou plusieurs matières biologiques; une deuxième membrane poreuse en communication fluidique avec la deuxième chambre; et une troisième chambre en communication fluidique avec la deuxième membrane poreuse, la troisième chambre comprenant une admission et une évacuation, et la troisième chambre étant fonctionnelle pour faire circuler un fluide biologique à travers l'admission et l'évacuation à un deuxième débit. La présente invention concerne également des procédés permettant de constituer des composants biologiques dans de tels systèmes.
PCT/US2024/058124 2023-12-05 2024-12-02 Organe sur puce multicouche dynamique (dynamoc) pour la simulation complète de la physiopathologie des organes Pending WO2025122436A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363606554P 2023-12-05 2023-12-05
US63/606,554 2023-12-05

Publications (1)

Publication Number Publication Date
WO2025122436A1 true WO2025122436A1 (fr) 2025-06-12

Family

ID=95980343

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/058124 Pending WO2025122436A1 (fr) 2023-12-05 2024-12-02 Organe sur puce multicouche dynamique (dynamoc) pour la simulation complète de la physiopathologie des organes

Country Status (1)

Country Link
WO (1) WO2025122436A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140335496A1 (en) * 2011-12-05 2014-11-13 Research Triangle Institute Human conducting airway model comprising multiple fluidic pathways
US20180291350A1 (en) * 2015-10-16 2018-10-11 Wake Forest University Health Sciences Multi-layer airway organoids and methods of making and using the same
US20200269234A1 (en) * 2018-02-23 2020-08-27 EMULATE, Inc. Microfluidic kidney-on-chip

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140335496A1 (en) * 2011-12-05 2014-11-13 Research Triangle Institute Human conducting airway model comprising multiple fluidic pathways
US20180291350A1 (en) * 2015-10-16 2018-10-11 Wake Forest University Health Sciences Multi-layer airway organoids and methods of making and using the same
US20200269234A1 (en) * 2018-02-23 2020-08-27 EMULATE, Inc. Microfluidic kidney-on-chip

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DONGEUN HUH, BENJAMIN D. MATTHEWS,AKIKO MAMMOTO,MARTíN MONTOYA-ZAVALA, HONG YUAN HSIN, DONALD E. INGBER: "Reconstituting Organ-Level Lung Functions on a Chip", SCIENCE, vol. 328, no. 5986, 25 June 2010 (2010-06-25), pages 1662 - 1668, XP055543104, DOI: 10.1126/science.1188302 *

Similar Documents

Publication Publication Date Title
Nahak et al. Advances in organ-on-a-chip materials and devices
Zhang et al. Organ-on-a-chip devices advance to market
Zhang et al. Advances in organ-on-a-chip engineering
Inamdar et al. Microfluidic cell culture models for tissue engineering
Sung et al. Microfabricated mammalian organ systems and their integration into models of whole animals and humans
Wang et al. Organ-on-a-chip platforms for drug delivery and cell characterization: A review
KR102067311B1 (ko) 개선된 수명 시간 및 항상성을 갖는 다기관 칩 장치
Yum et al. Physiologically relevant organs on chips
Tabatabaei Rezaei et al. Recent advances in organ‐on‐chips integrated with bioprinting technologies for drug screening
US8748180B2 (en) Microfluidic device for pharmacokinetic-pharmacodynamic study of drugs and uses thereof
JP5801311B2 (ja) 細胞培養用のマイクロスケール多流体流バイオリアクタ
US7776021B2 (en) Micromachined bilayer unit for filtration of small molecules
Valverde et al. Biomimetic models of the glomerulus
Park et al. 3D miniaturization of human organs for drug discovery
AVCI et al. Recent advances in organ-on-a-chip technologies and future challenges: a review
WO2016154319A1 (fr) Placenta artificiel et procédés de préparation
CN107980057A (zh) 用于体外3d细胞培养实验的微流控装置
Palaninathan et al. Multi-organ on a chip for personalized precision medicine
JP2017501745A (ja) 体外での複雑な生体組織の再構成のための流体デバイスおよび灌流システム
JP2017514524A (ja) 細胞層の配列のインビトロ産生方法およびデバイス
Das et al. An in-vivo-mimicking 3D lung cancer-on-a-chip model to study the effect of external stimulus on the progress and inhibition of cancer metastasis
Liu et al. Engineering in vitro vascular microsystems
CN114854588B (zh) 一种屏障-干细胞归巢仿生微流控芯片及其应用
WO2025122436A1 (fr) Organe sur puce multicouche dynamique (dynamoc) pour la simulation complète de la physiopathologie des organes
KR20230143372A (ko) 장기 모사 칩

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24901367

Country of ref document: EP

Kind code of ref document: A1