Dynamic multilayered organ-on-chip (dynamoc) for the comprehensive simulation of organ pathophysiology

Abstract

The present disclosure pertains to a microfluidic system that includes: 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; a first porous membrane in fluid communication with the first chamber; 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; a second porous membrane in fluid communication with the second chamber; and 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. The present disclosure also pertains to methods of forming biological components in such systems.

Description

TITLEDYNAMIC MULTILAYERED ORGAN-ON-CHIP (DYNAMOC) FOR THE COMPREHENSIVE SIMULATION OF ORGAN PATHOPHYSIOLOGYSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under 1452943 awarded by the National Science Foundation, and under R15 AI169564 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 606,554, filed on December 5, 2023. The entirety of the aforementioned application is incorporated herein by reference.BACKGROUND

[0003] A need exists for methods and systems to develop in vitro models that closely mimic the in vivo structure and function of tissues and organs. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to a microfluidic system. In some embodiments, 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.

[0005] Additional embodiments of the present disclosure pertain to methods of forming a biological component in a microfluidic system of the present disclosure. In some embodiments, 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.DRAWINGS

[0006] FIGS. 1A-1C provide illustrations of a microfluidic system in accordance with various embodiments of the present disclosure.

[0007] 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.

[0008] 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.

[0009] FIG. 4 is a schematic of a prototype of DynaMOC with molds and PDMS Chip Pieces.

[0010] FIG. 5 shows valve endothelial cells (VECs) cultured on a porous membrane and valve interstitial cells (VICs) cultured in a multilayered hydrogel. Scale bar = 100pm.

[0011] FIG. 6A is a wall shear stress profile of an oscillatory flow showing 10 dynes / cm2shear stress at a porous membrane.

[0012] 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.

[0013] FIG. 6C is a wall shear stress profile of the laminar flow showing 20 dynes / cm2shear stress at the porous membrane.

[0014] 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.

[0015] FIG. 8 shows a schematic representing the alternative design for a high throughput version of the Chip.

[0016] FIG. 9 shows a schematic representing the alternative designs for modifying the middle chamber.

[0017] FIG. 10 is a schematic where DynaMOC serves as a cross-section of a blood-brain barrier.

[0018] 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).

[0019] FIG. 12 shows additional variations of the microfluidic systems of the present disclosure.DETAILED DESCRIPTION

[0020] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components includes one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0021] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0022] A need exists for methods and systems to develop in vitro models that closely mimic the in vivo structure and function of tissues and organs. Such in vitro models can be utilized to evaluate various biological processes, such as disease mechanisms and disease progression.

[0023] For example, in the specific instance of the heart valve, 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.

[0024] However, current OOCs have numerous limitations in terms of mimicking actual organs. Moreover, current OOCs have limitations in terms of versatility for mimicking different types of organs.

[0025] In sum, a need exists for methods and systems to develop in vitro models that closely mimic the in vivo structure and function of tissues and organs. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0026] In some embodiments, the present disclosure pertains to a microfluidic system. With reference to FIGS. 1A-1C for illustrative purposes, 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.

[0027] 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.

[0028] 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.

[0029] Second chamber 18 is in fluid communication with the first porous membrane 16. In some embodiments, second chamber 18 is generally operable to serve as a reservoir for one or more biological materials. In some embodiments, second chamber 18 includes a plurality of compartments. In some embodiments, second chamber 18 includes at least two compartments 18A and 18B. In some embodiments, each compartment is operable to serve as a reservoir for one or more biological materials. In some embodiments, second chamber 18 is positioned between the first porous membrane 16 and the second porous membrane 20.

[0030] Second porous membrane 20 is in fluid communication with the second chamber 18 and the third chamber 22. In some embodiments, second porous membrane 20 includes a plurality of apertures 21 to facilitate fluid flow from second chamber 18 to third chamber 22. In some embodiments, second porous membrane 20 is between the third chamber 22 and the second chamber 18.

[0031] 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.

[0032] In some embodiments, 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.

[0033] Additional embodiments of the present disclosure pertain to methods of forming a biological component by utilizing the microfluidic systems of the present disclosure. With reference to FIGS. 1A-1C for illustrative purposes, 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. In some embodiments, such processes result in the formation of a biological component in the microfluidic system.

[0034] Biological fluids

[0035] 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. For instance, in some embodiments, the biological fluids include, without limitation, cell culture fluids, cells, cellular matrices, or combinations thereof.

[0036] Biological materials

[0037] 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. For instance, in some embodiments, the biological materials include, without limitation, hydrogels, cells, collagen, extracellular matrices, glycosaminoglycans, elastin, laminin, fibronectin, organoids, or combinations thereof. In some embodiments, the biological materials of the present disclosure can include combinations of different extracellular matrix components along with cells. For instance, in some embodiments (e.g., embodiments pertaining to heart valve simulation), the biological materials of the present disclosure can include valve interstitial cells in a matrix made with collagen, glycosaminoglycans, and elastin. In some embodiments, the biological materials of the present disclosure can also include organoids.

[0038] First and second flow rates

[0039] 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.

[0040] For instance, in some embodiments, 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.

[0041] First and second flow rates may be actuated in first and third chambers in various manners. For instance, in some embodiments, first and second flow rates may be actuated by one or more pumps. In some embodiments, the pumps include, without limitation, peristaltic pumps, syringe pumps, or combinations thereof.

[0042] Biological sensors

[0043] In some embodiments, the microfluidic systems of the present disclosure can also include one or more biological sensors that are operable to sense one or more conditions. In some embodiments, the methods of the present disclosure also include a step of sensing one or more conditions through one or more biological sensors.

[0044] The biological sensors of the present disclosure may be utilized to sense various conditions. For instance, in some embodiments, the conditions include, without limitation, pH, oxygen levels, lactic acid levels, glucose levels, electrical resistance, or combinations thereof.

[0045] Biological components

[0046] In some embodiments, 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. In some embodiments, the biological component is an organ, an organ component, a tissue, or combinations thereof. In some embodiments, 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. In some embodiments, the microfluidic system is operable to mimic the tissue structure, cellular components, and mechanical environment of the biological component.

[0047] In some embodiments, 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. In some embodiments, the biological component is a heart tissue. In some embodiments, the heart tissue mimics heart blood flow, contraction-relaxation of a beating heart, or combinations thereof.

[0048] In some embodiments, the biological component is a heart valve. In some embodiments, the biological fluids of the first chamber 12 and the third chamber 22 include valve endothelial cells (VEC). In some embodiments, the biological materials of the second chamber 18 include, without limitation, valve interstitial cells (VICs), collagen, glycosaminoglycans, or combinations thereof. In some embodiments, the first flow rate of the first chamber 12 includes an oscillatory flow rate to simulate an aortic side of an aortic valve. In some embodiments, the second flow rate of the third chamber 22 includes a steady flow rate to simulate a ventricular side of an aortic valve.

[0049] In some embodiments, 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. In some embodiments, second chamber 18 can include vascular smooth muscle cells and / or valve interstitial cells (VICs) to simulate the artery and / or valve. In some embodiments, 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.

[0050] In some embodiments, the biological component is a liver. In some embodiments, the first flow rate of the first chamber 12 is operable to simulate blood flow. In some embodiments, the biological fluids of the first chamber 12 include sinusoidal endothelial cells and Kupffer cells to mimic a vascular layer. In some embodiments, the biological materials of the second chamber 18 include, without limitation, stellate cells, hepatocytes, collagen, fibronectin, organoids, glycosaminoglycan, laminin, or combinations thereof. In some embodiments, the biological fluids of the third chamber 22 include hepatic cells, biliary endothelial cells, and cholangiocytes to mimic a bile duct. In some embodiments, the second flow rate of the third chamber 22 is operable to simulate bile duct flow.

[0051] In some embodiments, the biological component is a blood-brain barrier. In some embodiments, the third chamber 22 can serve as a vascular channel that includes brain microvascular endothelial cells along with fluid flow. In some embodiments, 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. In some embodiments, 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.

[0052] Compositions

[0053] The components of the microfluidic systems of the present disclosure may include various compositions. For instance, in some embodiments, the microfluidic system components include one or more elastic polymers. In some embodiments, the elastic polymers include polydimethylsiloxane (PDMS).

[0054] Advantages and Applications

[0055] The 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.

[0056] As such, the microfluidic systems and methods of the present disclosure can have numerous applications. For instance, in some embodiments, 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. In some embodiments, the microfluidic systems and methods of the present disclosure can be utilized to evaluate personalized medicine treatments. In some embodiments, the microfluidic systems and methods of the present disclosure can be utilized to evaluate long-term longitudinal imaging and assessment of cells.

[0057] In some embodiments, the microfluidic systems and methods of the present disclosure can be utilized to evaluate drug efficacy and / or drug toxicity. For instance, in some embodiments, 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).

[0058] Additional embodiments

[0059] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0060] Example 1. Development of a dynamic multi-layered organ-on-chip (DYNAMOC)

[0061] In this Example, Applicant describes the development of an organ-on-chip (OOC), a three-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.

[0062] 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. For example, in the specific instance of the heart valve, 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, 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.

[0063] Currently, there exists no OOCs that are able to mimic the complex tissue structure, cellular components, and mechanical environment of the heart valve (FIG. 2). Applicant’s OOC, called the dynamic multilayered organ on chip (DynaMOC), fills this 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. DynaMOC is thus a multilayered multiplex dynamic platform for a comprehensive simulation of organ pathophysiology.

[0064] 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.

[0065] 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. Similarly, 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.

[0066] 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).

[0067] 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.

[0068] For ease of assembly and cell seeding, 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.

[0069] For the specific application to study aortic valve, DynaMOC can serve as an aortic valve on chip (VOC) (FIG. 2). Additionally, 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 / cm2oscillatory 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 / cm2pulsatile 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.

[0070] To Applicant’s knowledge, there exists no single study model that can recapitulate all these key components such that their effect can be studied individually or simultaneously on the cells and disease progression. 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.

[0071] Additionally, such models can replace several small animal models and expensive in vitro alternatives that do not incorporate all key factors described herein. Moreover, DynaMOC can be used to assess several existing drugs and treatment strategies on patient-derived cells to further personalized medicines.

[0072] Example 1,1. DynaMOC design and assembly

[0073] 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.

[0074] To construct the PDMS chambers, 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.

[0075] To construct 1 DynaMOC, 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.

[0076] 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.

[0077] Example 1,2, Addition of co-culture and multilayered hydrogel

[0078] For the specific aortic valve application, the sterilized constructs are then seeded with valve endothelial cells (VECs) in the top and bottom flow chambers and interstitial cells in a hydrogel in the inner middle chamber (FIG. 2). After allowing the cells to grow and attach, 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.

[0079] 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) (Hyaluronic acid and Chondroitin sulfate) is used to mimic the spongiosa layer. First, collagen 1 is mixed with valve interstitial cells (VICs) and seeded onto the inner middle chamber. Once the hydrogel is solidified, Collagen 1 mixed with GAGs and VICs are added over the hydrogel to form the next layer. This method can be then utilized to add a further iteration of the hydrogel with elastin to mimic the ventricular! s layer.

[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.

[0081] Example 1.3. Utility and scope of DynaMOC

[0082] 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.

[0083] For diseased chips, 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.

[0084] Example 1,4, Compatible assays

[0085] 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.

[0086] Example 1,5. Versatility of Chip Application

[0087] DynaMOC can also be used to simulate other organ types, for example, kidney, and assess multi-organ diseases and comorbidities with aortic valve disease. Briefly, 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. It can also be used as a gut-on-chip with the top flow chamber as the intestinal channel, the middle chamber can be shortened for the matrix and the bottom flow chamber can be the vascular channel with endothelial and immune cells.

[0088] 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.

[0089] 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. Overall, 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.

[0090] While the entire system consists of 2 halves that can be put together to construct a single chip, it can also be used as a half for certain applications if needed. For example, as illustrated in FIG. 10, 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. In this scenario, the other half can serve as a lid / cover for the culture or as a media reservoir for the cells.

[0091] 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.

[0092] 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.

[0093] Additionally, the application of stretch, flow, and co-culture of different cell types including a hydrogel has never been implemented simultaneously in a platform, making it one of the most physiologically relevant organ-on-chip systems. This is the first chip where a multilayered hydrogel with VICs embedded in the gel and VECs seeded in a monolayer on top have been accommodated in a dynamic platform. Previous multilayered hydrogels for aortic valve applications have not utilized fully biotic hydrogels to create a multilayered structure with homogenously embedded VICs and a VEC monolayer in a dynamic environment.

[0094] Additionally, 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.

[0095] Presently, prototypes of DynaMOC have been constructed and tested for the seeding of cells and hydrogel (FIG. 5). Applicant has also tested that the middle chambers are able to close and attach. Computational fluid dynamics simulations have been used to validate that physiological oscillatory shear and laminar shear stresses can be applied to the endothelial cells in the flow chambers (FIGS. 6A-6C).

[0096] Current plans include testing the strain translation of the applied stretch to the hydrogel. Preliminary versions of the chip have been used to ensure stain translation on the porous membranes (FIG. 7). Preliminary versions of DynaMOC have also been used to ensure that the stretch and flow can be applied simultaneously on the chip and cells and that hydrogels can be seeded on the chip.

[0097] Given the versatility of the design, DynaMOC ean be used to seed cells from and mimic any organ. 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.

[0098] 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.

[0099] Example 1,6. Dynamic Longitudinal Assessments

[0100] Given that the chip is constructed using PDMS, a self-healing, flexible polymer, and the chip has multiple compartments, 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.). The incorporation of a bioelectrochemical sensing module can be facilitated with this chip.

[0101] Example 1.7. Size Alterations

[0102] The current dimensions of 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.

[0103] Example 1.8. High Throughput Chip

[0104] Additionally, 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).

[0105] Example 1,9. Middle Chamber Modifications

[0106] The middle chamber itself can be further divided into multiple middle chambers, either horizontally or vertically, to assemble more complex extracellular matrices (FIG. 9). To facilitate that 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. For example, 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.

[0107] Example 1,10. Top and Bottom Flow Chamber Modifications

[0108] As shown in FIGS. 11A-11B, 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. In certain applications, where fluid flow is not needed, 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. Additionally, as shown in FIG. 11C, the inlet and outlet ports can also be placed on an angle to accommodate specific and unique flow needs.

[0109] Furthermore, as shown in FIG. 12, 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.

[0110] Example 1,11. Summary

[0111] Organ-on-chip (OOC) 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. Currently, there exist no OOCs that are able to mimic the complex tissue structure, cellular components, and mechanical environment of complex multilayered, dynamic tissues.

[0112] 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.

[0113] 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. To Applicant’s knowledge, there exists no single study model that can recapitulate all these key components such that their effect can be studied individually or simultaneously on the cells and disease progression.

[0114] 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.

[0115] Overall, 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.

[0116] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein

Claims

CLAIMS:

1. A microfluidic system comprising: a first chamber comprising an inlet and an outlet, wherein the first chamber is operable to flow a biological fluid through the inlet and the outlet at a first flow rate; a first porous membrane in fluid communication with the first chamber; a second chamber in fluid communication with the first porous membrane, wherein the second chamber is operable to serve as a reservoir for one or more biological materials; a second porous membrane in fluid communication with the second chamber; and a third chamber in fluid communication with the second porous membrane, wherein the third chamber comprises an inlet and an outlet, and wherein the third chamber is operable to flow a biological fluid through the inlet and the outlet at a second flow rate.

2. The microfluidic system of claim 1, wherein the biological fluid that is operable to flow through each of the first chamber and the third chamber is selected from the group consisting of cell culture fluids, cells, cellular matrices, or combinations thereof.

3. The microfluidic system of claim 1, wherein the biological materials are selected from the group consisting of hydrogels, cells, collagen, extracellular matrices, glycosaminoglycans, elastin, laminin, fibronectin, organoids, or combinations thereof.

4. The microfluidic system of claim 1, wherein the second chamber is positioned between the first porous membrane and the second porous membrane.

5. The microfluidic system of claim 1, wherein the second porous membrane is between the third chamber and the second chamber.

6. The microfluidic system of claim 1, wherein the first porous membrane is between the first chamber and the second chamber.

7. The microfluidic system of claim 1, wherein the second chamber comprises a plurality of compartments, wherein each compartment is operable to serve as a reservoir for one or more biological materials.

8. The microfluidic system of claim 1, wherein the second chamber comprises at least two compartments.

9. The microfluidic system of claim 1, further comprising one or more biological sensors operable to sense one or more conditions.

10. The microfluidic system of claim 9, wherein the one or more conditions arc selected from the group consisting of pH, oxygen levels, lactic acid levels, glucose levels, electrical resistance, or combinations thereof.

11. The microfluidic system of claim 1, wherein the microfluidic system is operable to form a biological component within the system.

12. The microfluidic system of claim 11, wherein the biological component is an organ, an organ component, a tissue, or combinations thereof.

13. The microfluidic device of claim 1, wherein the inlet and the outlet of the first chamber are in lateral positions.

14. The microfluidic device of claim 1, wherein the inlet and the outlet of the first chamber are in angled positions.

15. The microfluidic device of claim 1, wherein the inlet and the outlet of the third chamber are in lateral positions.

16. The microfluidic device of claim 1, wherein the inlet and the outlet of the third chamber are in angled positions.

17. A method of forming a biological component in a microfluidic system, wherein the microfluidic system comprises: a first chamber comprising an inlet and an outlet, a first porous membrane in fluid communication with the first chamber, a second chamber in fluid communication with the first porous membrane, a second porous membrane in fluid communication with the second chamber, and a third chamber in fluid communication with the second porous membrane and comprising an inlet and an outlet; and wherein the method comprises: flowing a biological fluid through the inlet and the outlet of the first chamber at a first flow rate, placing one or more biological materials in the second chamber, and flowing a biological fluid through the inlet and the outlet of the third chamber at a second flow rate.

18. The method of claim 17, wherein the biological fluid flowing through each of the first chamber and the third chamber is selected from the group consisting of cell culture fluids, cells, cellular matrices, or combinations thereof.

19. The method of claim 17, wherein the biological materials are selected from the group consisting of hydrogels, cells, collagen, extracellular matrices, glycosaminoglycans, elastin, laminin, fibronectin, organoids, or combinations thereof.

20. The method of claim 17, wherein the first flow rate of the first chamber and the second flow rate of the third chamber represent the same flow rates.

21. The method of claim 17, wherein the first flow rate of the first chamber and the second flow rate of the third chamber represent different flow rates.

22. The method of claim 17, wherein one of the first or second flow rates comprises an oscillatory flow rate, and wherein the other of the first or second flow rates comprises a steady flow rate.

23. The method of claim 17, wherein at least one of the first or second flow rates mimics fluid shear stress.

24. The method of claim 17, further comprising sensing one or more conditions through one or more biological sensors.

25. The method of claim 24, wherein the one or more conditions arc selected from the group consisting of pH, oxygen levels, lactic acid levels, glucose levels, electrical resistance, or combinations thereof.

26. The method of claim 17, wherein the biological component is an organ, an organ component, a tissue, or combinations thereof.

27. The method of claim 17, wherein 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.

28. The method of claim 17, wherein the biological component is a heart valve, wherein the biological fluids of the first chamber and third chamber comprise valve endothelial cells (VEC), wherein the biological materials of the second chamber are selected from the group consisting of valve interstitial cells (VICs), collagen, glycosaminoglycans, or combinations thereof,wherein the first flow rate of the first chamber comprises an oscillatory flow rate to simulate an aortic side of an aortic valve, and wherein the second flow rate of the third chamber comprises a steady flow rate to simulate a ventricular side of an aortic valve.

29. The method of claim 17, wherein the biological component is a liver, wherein the first flow rate of the first chamber is operable to simulate blood flow, wherein the biological fluids of the first chamber comprise sinusoidal endothelial cells and Kupffer cells to mimic a vascular layer, wherein the biological materials of the second chamber are selected from the group consisting of stellate cells, hepatocytes, collagen, fibronectin, glycosaminoglycan, laminin, or combinations thereof, wherein the biological fluids of the third chamber comprise hepatic cells, biliary endothelial cells, and cholangiocytes to mimic a bile duct, and wherein the second flow rate of the third chamber is operable to simulate bile duct flow.

30. The method of claim 17, wherein the biological component is a blood-brain barrier, wherein the third chamber serves as a vascular channel comprising brain microvascular endothelial cells along with fluid flow, wherein at least half of the second chamber serves as a neural chamber comprising a sequential assembly of pericytes, astrocytes, microglia, and neurons in an extracellular matrix, and wherein at least another half of the second chamber serves as a lid, a cover, a media reservoir for the cells, or combinations thereof.

31. The method of claim 17, wherein the method is utilized to evaluate disease mechanisms, disease progression, treatment strategics, immune responses, infectious and degenerative diseases, co-morbidities, drug efficacy, drug toxicity, or combinations thereof.

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