Microfluidic gut-liver integrated chip

Abstract

Embodiments are generally directed to a microfluidic device comprising liver and gut physiologies, wherein a gut physiology and a liver physiology are fluidly connected by a vascular system divided into a gut vascular chamber and a liver vascular chamber by a porous micropillar membrane structure.

Description

MICROFLUIDIC GUT-LIVER INTEGRATED CHIPCross Reference to Related Application

[0001] The present application claims priority to U.S. provisional Application No. 63 / 507,682 entitled “MICROFLUIDIC GUT-LIVER INTEGRATED CHIP”, filed on June 12, 2023. The entire contents of the above-listed application are hereby incorporated by reference for all purposes.Technical Field

[0002] The present disclosure pertains generally to a physiology-on-a-chip microfluidic device which integrates gut and liver physiology on a single chip.Government Support Clause

[0003] This invention was made with government support under Grant Number 1728407 awarded by the National Science Foundation. The government has certain rights in the invention.Background and Summary

[0004] Both gut and liver tissues serve important physiological functions. One key function of the gut and liver is the metabolism of food and xenobiotics. The first prominent digesting organ is the gut where a rich microbial system in combination with enterocytes decomposes the ingested substrates. The substrates are then absorbed through the intestine wall and travel to the liver where they engage in hepatic metabolism. This combined digestive action determines the metabolic pathways of most substrates and chemicals.

[0005] One significant implication of this digestive power is in drug discovery. In drug design, drug candidates may have good drug-likeness but the digestion of the drug by the gut and liver, known as the first-pass metabolism, reduces the drug activity to such an extent that only a small amount of active drug reaches the target organ through the circulatory system. Currently, the most reliable means to estimate the drug response is animal testing, but this technique is highly costly considering the large number of drug candidates. Additionally, animal testing sufferers from infidelity between human and animal physiology, and is associated with ethical issues.

[0006] Embodiments of the disclosed technology are generally directed to a novel in-vitro microfluidic device that mimics the physiology of the gut and liver organs on a compartmented single-chip device. A microfluidic device as further described herein comprises liver and gut physiologies, wherein a gut physiology and a liver physiology are fluidly connected by a vascular system divided into a gut vascular chamber and a liver vascular chamber by a porous micropillar membrane structure.

[0007] It should be understood that the summary above is provided to introduce in simplified form a selection of concepts that are further described in the detailed description. It is not meant to identify key or essential features of the claimed subject matter, the scope of which is defined uniquely by the claims that follow the detailed description. Furthermore, the claimed subject matter is not limited to implementations that solve any disadvantages noted above or in any part of this disclosure.Brief Description of the Drawings

[0008] The present disclosure will be better understood from reading the following description of non-limiting embodiments, with reference to the attached drawings, wherein below:

[0009] FIG. 1 illustrates a side cross-section view of an example microfluidic gut-liver device in accordance with certain embodiments of the disclosed technology;

[0010] FIG. 2 illustrates a top view of an example of each of three layers of the gut-liver device in accordance with certain embodiments of the disclosed technology;

[0011] FIG. 3 illustrates a perspective view of each of three layers of the gut-liver device in accordance with certain embodiments of the disclosed technology; and

[0012] FIG. 4 illustrates an example of a cellular composition of the gut-liver chip in accordance with certain embodiments of the disclosed technology.Detailed Description

[0013] Certain embodiments of the disclosed technology are directed to a compact, microfluidic gut-liver integrated chip comprising gut and liver physiologies. The herein described “gut-liver chip” (also referred to herein as “the device”) mimics interactions between the gut and the liver occurring through the vascular system. A vascular system included in the gut-liver chip is flexible to model the lamina propria (the vascular section in contact with the gut, lined withvascular endothelial cells), the portal vain (the vascular connection between the intestine and the liver), and the vascular lumen within the liver (covered with liver sinusoidal endothelial cells).

[0014] The gut-liver chip may be modular to include various cellular aspects of the gut and liver domains, including the microbiome and the liver lobule. Due to a rigorous representation of the gut-liver axis, the gut-liver chip may be used for a variety of physiological and pathological studies, including drug discovery. For example, the device aims to provide a probing tool to reduce the number of animal trials.

[0015] Benefiting from a modularized structure, the gut-liver chip offers a new way to incorporate gut physiology and microbiology, the blood circulatory system between the gut and liver including the portal vein, and the liver physiology and function. Various cellar populations can be cultured and studied simultaneously in the device, including but not limited to the gut microbiome, the gut epithelial and endothelial tissues, immune cells, and the hepatic cellular system including hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs), all on one chip.

[0016] Leveraging the microfluid capabilities, the device further allows the simulation of the microenvironment containing mechanical and chemical cues around cellar populations which makes it possible to recapitulate the interaction between cells and tissues along the gut-liver axis. While addressing the complexity of the gut-liver axis, the device is conceptually simple and easy to operate, making it suitable for commercial and laboratory use.

[0017] The device can address this challenge by providing an inexpensive high-throughput screening platform for use prior to animal testing. This can significantly reduce the drug research cost for a large number of drugs. Another application of the device is modeling various multiorgan diseases that involve the gut and liver. An example of such a disease is the non-alcoholic fatty liver disease (NAFLD), a largely untreatable disease that inflicts more than 25% of the population. The on-chip simulation of the diseases could facilitate pathological research and the discovery of therapeutics.

[0018] While various designs of gut-on-a-chip and liver-on-a-chip devices have been developed, there has been little progress on the integration of the gut and liver physiologies on chips (e.g., a single chip) and a few existing composite configurations have serious limitations. Namely, the most common drawback of single-chip designs is their oversimplicity. For instance, some hybrid gut-liver chips consist of a top layer to culture the gut epithelium and a bottom layerto culture the liver. In this scheme, the liver model is unrigorous, as key aspects such as hepatic sinusoidal endothelial cells and separate bile and blood from the hepatocytes are overlooked. This detracts from the device's functionality in many applications (e.g., in the drug design the absorption rate of the drug would be inaccurate).

[0019] Another common type of composite platform is based on the human-on-a-chip concept. Human-on-a-chip usually involves microfluidic fabrication of various chambers each representing a specific organ. Whether based on a multi-chip or single-chip device, these devices typically require multiple macroscale fluidic connections or prime movers to interconnect these organ chambers. The heavy reliance on peripheral equipment and incompact designs makes these devices inefficient to operate and use. The device complexity also hinders the physiologically-relevant incorporation of multiple cellular subcultures. These limitations are of double importance in the case of the gut and liver because these organs feature diverse cellular populations and complex physiology.

[0020] Embodiments of the disclosed technology offer a compact single-chip solution for creating a combined gut and liver model. While based on a triple-layer design concept and hence bearing no significant fabrication effort relative to the previously single-chip models, it is more physiologically relevant due to the combined modeling of the gut, the liver, and vascular systems. Moreover, the vascular system is uniquely divided into two compartments by a porous micropillar membrane structure to allow for customized cell cultures for the gut and the liver.

[0021] The device, as a result, requires minimum fluidic connections and external fluid drivers, making it more user-friendly than human chips. In addition, the use of dedicated ports for tissue growth and the normal operation phases in segregated chambers enables culturing of various cell types which is absent in the human chips. This device is, thus, the first to support the intimate coculture of the gut microbiome, the gut epithelial and endothelial tissues, immune cells, hepatocytes hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.

[0022] FIG. 1 illustrates a cross-sectional side view of a gut-liver chip 100 in accordance with certain embodiments of the disclosed technology. The gut-liver chip 100 comprises a gut chamber 1 which is fluidly coupled to a gut vascular chamber 3. Gut chamber ports 8 extends from the gut chamber 1, as further described with respect to FIG. 2. A vascular blood inlet 6 and gut vascular culture ports 7 extend from the gut vascular chamber 3 and couple the gut vascular chamber 3 and the gut chamber 1 to an environment outside the gut-liver chip 100. The gut-liver chip 100 furthercomprises a liver chamber 2 which is fluidly coupled to a liver vascular chamber 4. Liver chamber ports 11 extend from the liver chamber 2, as further described with respect to FIG. 2.

[0023] A vascular blood outlet 9 and liver vascular culture ports 10 extend from the liver vascular chamber 4 and couple the liver vascular chamber 4 and the liver chamber 2 to an environment outside the gut-liver chip 100. The gut chamber 1 and the liver chamber 2 are separate from each other.

[0024] A porous micropillar membrane structure 5 splits a vascular system into two subsections, each associated with either gut or the liver. For example, the gut vascular chamber 3 and the liver vascular chamber 4 are subsections of the vascular system of the gut-liver chip 100, and a porocity of the porous micropillar membrane structure 5 enables fluidic communication between the two subsections. This division allows for culturing a separate vascular system for each organ which is a novel feature of the device to mimic the in-vivo condition.

[0025] The gut chamber 1 and the liver chamber 2 are positioned in a top layer of the gut-liver chip 100, and the gut vascular chamber 3 and the liver vascular chamber 4 are positioned in a bottom layer of the gut-liver chip 100, as further described with respect to FIG. 2

[0026] FIG. 2 shows a top-down view 200 of each of three layers 302, 304, 306 of the gutliver chip 100, including a top layer 304, a middle layer 302, and a bottom layer 306 in accordance with certain embodiments of the disclosed technology. FIG. 3 shows a perspective view 300 of each of the three layers 302, 304, 306 of the gut-liver chip 100 shown in FIG. 2 in accordance with certain embodiments of the disclosed technology. FIGS. 2 and 3 are described simultaneously herein.

[0027] The gut-liver chip 100 has a compact modularized design that recapitulates the key physiological function of the gut-liver axis in three interconnected layers. The top layer 304 houses the gut and liver models (e.g., the gut chamber 1 and the liver chamber 2) as separate compartments. Each of the gut and liver chambers have at least one inlet and one outlet port to introduce cellular cultures during tissue growth and normal operation. The gut chamber ports 8 of the gut chamber 1 as described with respect to FIG. 1 include a gut chamber inlet Gi and a gut chamber outlet Go. The liver chamber ports 11 of the liver chamber 2 as described with respect to FIG. 1 include a liver chamber inlet Li and a liver chamber outlet Lo.

[0028] The bottom layer 306 embeds the circulatory system (e.g., the vascular system comprised of the gut vascular chamber 3 and the liver vascular chamber 4) that connects the liverphysiology and the gut physiology (e.g., the liver chamber 2 and the gut chamber 1). Each vascular division has at least three ports; two ports to introduce and remove the cellular culture for tissue growth and a third port to inject the “blood” between the gut and liver during the normal operation. As described with respect to FIG. 1, the gut vascular chamber 3 comprises the vascular blood inlet 6 (VGi-2) and gut vascular culture ports 7.

[0029] The gut vascular culture ports 7 include a gut vascular culture inlet VGi-i and a gut vascular culture outlet VGo. As described with respect to FIG. 1, the liver vascular chamber 4 comprises the vascular blood outlet 9 (LG0-2) and liver vascular culture ports 10. The liver vascular culture ports 10 include a liver vascular culture inlet LGi and a liver vascular culture outlet LG0-1.

[0030] The middle layer 302 comprises a porous membrane that supports cell culture on both sides while maintaining a physical connection between the organs and the vascular system. For example, the porous membrane has a first side in contact with the top layer / the liver chamber 2 and the gut chamber 1, and a second side in contact with the bottom layer / the gut vascular chamber 3 and the liver vascular chamber 4, where the first side and the second side both support cell culture, as further described with respect to FIG. 4.

[0031] The porous membrane of the middle layer may have a first thickness 302 which is thin (e.g., 50 pm) to allow efficient communication between cells on either side. A second thickness 304 and a third thickness 306 of each of the top layer and the bottom layer respectively may be within a thickness range spanning a few millimeters (e.g., 1 -3mm), which may allow easy handling of the device. In the case of the need to perform real-time microscopy, either the top or bottom layer could be made thinner to enable higher-quality observations.

[0032] The device can be fabricated using the well-known soft lithography technique using polydimethylsiloxane (PDMS) as the base material. Each layer is fabricated separately and then all layers are bound together upon plasma treatment. The device assembly is finalized by punching ports that integrate the microdevice with the macro world comprised of fluidic connection, prime movers, and the liquid reservoirs (e.g., a typical assembly would include a syringe pump filled with a liquid solution or pressurized containers plugged into a pressurized nitrogen tank, with valves on the inlet stream).

[0033] Turning to FIG. 4, an example of a cellular composition 400 of the gut-liver chip 100 is shown. The system can grow various cell cultures during the “tissue growth” phase, as shown in FIG. 4. The simplest demonstration is comprised of the gut epithelium and hepatic cells. Thecellular composition 400 includes a gut microbiome 1, a gut epithelium 2, a gut endothelium 3, gut immune cells 4, hepatocytes 5, and liver sinusoidal endothelial cells 6.

[0034] Epithelial cells are introduced into the gut chamber to create a tissue layer on top of the porous membrane coated with an ECM matrix. It usually takes a few days (3-4) to achieve confluency, while the extended culture of up to 7-9 days’ results in the formation of villi-like protrusions resembling the gut topography. The application of mechanical pulsation further accelerates villi formation. The mechanical stimuli can be exerted by either pumping the cell culture using a peristaltic pump or applying fluctuating air pressure through lateral air chambers (not shown in the sketches) as previously shown in the single gut chips.

[0035] The hepatic cell culture can be similarly introduced into the ECM-coated liver chamber to establish a tissue. The hepatic tissue usually takes a few days (3-4) to grow to uniformity resulting in the device baseline model. The device base design can be further enhanced by including additional cell types. In one variation, hematopoietic stem cells (HSCs) can be cultured into the liver chamber before the hepatocytes. In another variation, after the cell culture is established in the top layer, the device is flipped, and the vascular cell culture is seeded into the bottom sub chambers to form endothelial tissues.

[0036] Immune cells and KCs can also be added to vascular chambers. Another common variation would include the addition of the microbiome in the gut chamber after all mammalian cell cultures. The entire tissue growth phase usually takes 1-2 weeks but can be accomplished in parallel for multiple devices to enhance the throughput.

[0037] Following the tissue growth, the physiological tissue environment can be simulated in the device’ s “normal operation” phase for each case study. For the top layer, the valves on the inlet could be switched to fresh streams, and, for the bottom layer, the tissue culture valves are closed, and the main inlet and outlet valves are opened to establish a fresh "blood" flow from the gut to the liver. The continuous injection of the nutrients in all chambers keeps the cells alive for at least a few weeks making it possible to run a variety of case studies. For drug testing and discovery research, the gut inlet streams could be mixed with the drug at the predefined concentration. Subsequent analysis of the drug concentration in the blood and liver provides important information about the candidates' first-pass metabolism.

[0038] While using immortal cell lines could be used for quick explorations, stem cells and native microbiomes can be employed as tissue to customize the device for each patient. Concerningdisease studies, various pathological effects can be simulated. NAFLD, for instance, could be simulated by injecting lipids into the gut inlet which over time results in the absorption of the fatty acids into the blood and triggering steatosis in the liver.

[0039] The integration of the gut and liver allows an in-depth study of various pathways related to NAFLD that could be originated from the gut-liver axis. For example, gut dysbiosis is widely known as a key etiological factor in the initiation and progression of NAFLD, however, the exact correlation between microbial signature and NAFLD is not yet known. The use of this platform can help to test and recreate various microbial compositions and find insight into the disease mechanism and potential therapeutics.

[0040] In an example, a microfluidic device can include: a liver physiology; and a gut physiology, wherein the gut physiology and the liver physiology are fluidly connected by a vascular system divided into a gut vascular chamber and a liver vascular chamber by a porous micropillar membrane structure.

[0041] The microfluidic device can be formed of: a top layer housing the gut physiology in a gut chamber and the liver physiology in a liver chamber separate from the gut chamber.

[0042] The microfluidic device can be further formed of: a middle layer comprising a porous membrane which provides a physical connection for each of the gut chamber and the liver chamber with the vascular system.

[0043] The microfluidic device can be further formed of: a bottom layer housing the vascular system, wherein each of the top layer, the middle layer, and the bottom layer are interconnected with each other.

[0044] In certain embodiments, the porous membrane of the middle layer is configured to support a cell culture on each of a first side in communication with the gut chamber and the liver chamber, and a second side opposite the first side and in communication with the gut vascular chamber and the liver vascular chamber.

[0045] In certain embodiments, the microfluidic device further includes a gut chamber inlet and a gut chamber outlet of the gut chamber, and a liver chamber inlet and a liver chamber outlet of the liver chamber.

[0046] In certain embodiments, the microfluidic device further includes a gut vascular culture inlet fluidly coupled to the gut vascular chamber.

[0047] In certain embodiments, the microfluidic device further includes a vascular blood inlet fluidly coupled to the gut vascular chamber.

[0048] In certain embodiments, the microfluidic device further includes a gut vascular culture outlet fluidly coupled to the gut vascular chamber.

[0049] In certain embodiments, the microfluidic device further includes a liver vascular culture inlet fluidly coupled to the liver vascular chamber.

[0050] In certain embodiments, the microfluidic device further includes a vascular blood outlet fluidly coupled to the liver vascular chamber.

[0051] In certain embodiments, the microfluidic device further includes a liver vascular culture outlet fluidly coupled to the liver vascular chamber.

[0052] In certain embodiments, the microfluidic device further includes at least one additional physiology fluidly connected to the gut physiology via the vascular system.

[0053] In certain embodiments, the at least one additional physiology is a skin physiology.

[0054] In certain embodiments, the at least one additional physiology is a kidney physiology.

[0055] In certain embodiments, the microfluidic device further includes: a gut vascular culture inlet fluidly coupled to the gut vascular chamber; a vascular blood inlet fluidly coupled to the gut vascular chamber; a gut vascular culture outlet fluidly coupled to the gut vascular chamber; a liver vascular culture inlet fluidly coupled to the liver vascular chamber; a vascular blood outlet fluidly coupled to the liver vascular chamber; and a liver vascular culture outlet fluidly coupled to the liver vascular chamber.

[0056] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0057] The previously described versions of the disclosed subject matter have many advantages that were either described or would be apparent to a person of ordinary skill. Even so,these advantages or features are not required in all versions of the disclosed apparatus, systems, or methods.

[0058] Additionally, this written description makes reference to particular features. It is to be understood that the disclosure in this specification includes all possible combinations of those particular features. Where a particular feature is disclosed in the context of a particular aspect or example, that feature can also be used, to the extent possible, in the context of other aspects and examples.

[0059] Also, when reference is made in this application to a method having two or more defined steps or operations, the defined steps or operations can be carried out in any order or simultaneously, unless the context excludes those possibilities.

[0060] Although specific examples of the invention have been illustrated and described for purposes of illustration, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

Claims

Claims1. A microfluidic device, comprising: a liver physiology; and a gut physiology, wherein the gut physiology and the liver physiology are fluidly connected by a vascular system divided into a gut vascular chamber and a liver vascular chamber by a porous micropillar membrane structure.

2. The microfluidic device of claim 1, wherein the microfluidic device is formed of: a top layer housing the gut physiology in a gut chamber and the liver physiology in a liver chamber separate from the gut chamber.

3. The microfluidic device of claim 2, wherein the microfluidic device is further formed of: a middle layer comprising a porous membrane which provides a physical connection for each of the gut chamber and the liver chamber with the vascular system.

4. The microfluidic device of claim 3, wherein the microfluidic device is further formed of: a bottom layer housing the vascular system, wherein each of the top layer, the middle layer, and the bottom layer are interconnected with each other.

5. The microfluidic device of claim 4, wherein: the porous membrane of the middle layer is configured to support a cell culture on each of a first side in communication with the gut chamber and the liver chamber, and a second side opposite the first side and in communication with the gut vascular chamber and the liver vascular chamber.

6. The microfluidic device of claim 4, further comprising: a gut chamber inlet and a gut chamber outlet of the gut chamber, and a liver chamber inlet and a liver chamber outlet of the liver chamber.

7. The microfluidic device of claim 4, further comprising: a gut vascular culture inlet fluidly coupled to the gut vascular chamber.

8. The microfluidic device of claim 7, further comprising: a vascular blood inlet fluidly coupled to the gut vascular chamber.

9. The microfluidic device of claim 8, further comprising: a gut vascular culture outlet fluidly coupled to the gut vascular chamber.

10. The microfluidic device of claim 4, further comprising: a liver vascular culture inlet fluidly coupled to the liver vascular chamber.

11. The microfluidic device of claim 10, further comprising: a vascular blood outlet fluidly coupled to the liver vascular chamber.

12. The microfluidic device of claim 11, further comprising: a liver vascular culture outlet fluidly coupled to the liver vascular chamber.

13. The microfluidic device of claim 1, further comprising: at least one additional physiology fluidly connected to the gut physiology via the vascular system.

14. The microfluidic device of claim 13, wherein: the at least one additional physiology is a skin physiology.

15. The microfluidic device of claim 13, wherein: the at least one additional physiology is a kidney physiology.

16. The microfluidic device of claim 4, further comprising: a gut vascular culture inlet fluidly coupled to the gut vascular chamber; a vascular blood inlet fluidly coupled to the gut vascular chamber; a gut vascular culture outlet fluidly coupled to the gut vascular chamber; a liver vascular culture inlet fluidly coupled to the liver vascular chamber;a vascular blood outlet fluidly coupled to the liver vascular chamber; and a liver vascular culture outlet fluidly coupled to the liver vascular chamber.

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