Microfluidic device

Abstract

The present invention relates to a microfluidic device, a microfluidic system including the microfluidic device, and a method for culturing biological substances.

Description

Technical Field

[0001] The present invention relates to a microfluidic device, a microfluidic system including the microfluidic device, and a method for culturing a biological substance.

[0002] The present invention generally relates to the field of cell biology or molecular biology, and particularly to the field of microfluidics. More specifically, the present invention relates to a biochip using microfluidic technology.

Background Art

[0003] A biochip is an engineered substrate ("miniaturized laboratory") capable of performing a number of biochemical reactions simultaneously in the field of molecular biology. One of the goals of biochip technology is to efficiently screen a number of biological analytes, and it can be applied in a wide range from disease diagnosis to detection of biological weapons.

[0004] An organ-on-a-chip (OOC) is a multi-channel three-dimensional microfluidic cell culture integrated circuit (chip) that simulates the activities, mechanisms, and physiological responses of tissues, whole organs, or organ systems, and is a type of artificial organ. The organ-on-a-chip is important as an issue in biomedical engineering research. The organ-on-a-chip is consistent with the purpose of manufacturing a microfluidic device including a substructure of a living artificial organ in a controlled microenvironment or nanoenvironment, and in real-time monitoring, it can reproduce various aspects of the dynamic behavior, functionality, and (pathological) physiological responses of organs in vivo. The fusion of labs-on-chip (LOC) and cell biology has enabled organ-specific human physiological research, and a new in vitro model of the human body composed of a large number of cells has been developed.

[0005] In multiple documents, there have been reports that the functions of organs could be transferred to such organ chips, but the application of microfluidic technology is still in its initial stage. The designs and methods of organ chips vary widely among researchers. Therefore, it is considered that long-term studies are required for the verification and optimization of microfluidic systems. Organs for which simulations are being conducted using microfluidic devices include the brain, lungs, heart, kidneys, liver, prostate, blood vessels (arteries), skin, bone, cartilage, and others.

[0006] However, for the construction of proper artificial organs, it is necessary not only to accurately manipulate cells but also to understand in detail the basic and complex responses of the human body to certain events. A common concern regarding organ chips is that the organs under test are isolated. Since the human body is a complex network of physiological processes, it is difficult to simulate using only a single organ.

[0007] Rennert et al. (2015) disclosed a biochip for simulating and analyzing human liver function in vitro in "A microfluidically perfused three dimensional human liver model, Biomaterials, Vol. 71, p. 119-131". Rennert et al. reported the establishment of three-dimensional liver organoids embedded in a microfluidically supported biochip, which is considered to have been inspired by the structure from the morphology of the liver.

[0008] Rogal et al. (2020) reported on the development of an organ chip integrating functionally mature human white adipocytes in "WAT-on-a-chip integrating human mature white adipocytes for mechanistic research and pharmaceutical applications, Scientific Reports, Vol. 10, p. 1-12". This known chip is a multi-layer device featuring a tissue chamber specially designed for the maintenance of three-dimensional tissues based on human primary adipocytes, with nutrients supplied through a perfusion media flow path.

[0009] Hori et al. (2019) disclosed a compact fluidic system in "Compact fluidic system for functional assessment of pancreatic islets, Biomedical Microdevices, Vol. 21, p. 1-9", which is considered to enable the ex vivo assessment of islet function for efficient islet transplantation. This known fluidic system comprises a chip embedded with a micromesh sheet. Islets can be placed on this micromesh sheet and observed under a microscope. Hori et al. reported that due to the high porosity of the micromesh sheet, the islets on the mesh sheet hardly moved during perfusion and were not damaged by hydraulic pressure. This known fluidic system was assembled using a sample fractionation chip of polydimethylsiloxane. This chip has flow paths and columns, and all surfaces are super-hydrophilic treated so that the solution flows through the chip by gravity.

[0010] Nourmohammadzadeh et al. (2013) disclosed a microfluidic pancreatic islet array based on hydrodynamic capture principles in "Microfluidic array with integrated oxygenation control for real-time live-cell imaging: effect of hypoxia on physiology of microencapsulated pancreatic islets, Analytical Chemistry, Vol. 85, p. 11240-11249". According to Nourmohammadzadeh et al., this array can be used for lab-on-a-chip research using live cell multi-parameter imaging, and this research is considered to enable the elucidation of physiological and pathophysiological changes in microencapsulated pancreatic islets in a hypoxic state.

[0011] Zbinden et al. (2020) disclosed a pancreatic endocrine gland chip model based on a customized microfluidic platform in "Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model, Matrix Biology, Vol. 85-86, P. 205-220", and this model enables self-guided capture of single human pseudo-islets. Summary of the Invention Problems to be Solved by the Invention

[0012] Current biochips used in the art utilize auxiliary structures, and in vitro modeling of the complex microenvironment of living organs such as the pancreas is still impossible. In particular, in known biochips, it is not possible to localize or immobilize artificial organs in a targeted manner in the limited space within the chip, and furthermore, culturing and analysis under physiological conditions cannot be performed, so in situ analysis of artificial organs cannot be carried out. Therefore, an object of the present invention is to provide a device that can avoid or at least reduce the drawbacks of the prior art. In particular, it is an object of the present invention to provide a microfluidic device that arranges an artificial organ structure in a physiological environment and enables its in situ analysis.

[0013] The present invention meets such needs and other needs.

Means for Solving the Problems

[0014] An object of the present invention is a microfluidic device comprising a first microfluidic channel, a second microfluidic channel, and a porous membrane, at least a part of the first microfluidic channel and the second microfluidic channel being separated by the porous membrane, the porous membrane having a capture structure configured to immobilize a biological substance at a predetermined position, the microfluidic device which can be solved by providing.

[0015] As used herein, the term "microfluidics" relates to a component in which a moving fluid is confined within one or more flow channels having one or more dimensions of 1 mm or less (microscale), or through which a moving fluid is guided through one or more flow channels of such dimensions. The microfluidic flow channels may be larger than the microscale in one or more directions, but are microscale in at least one direction. In some examples, the shape of the microfluidic flow channels may be configured to control the flow rate of the fluid passing through the channels (e.g., the height of the channels may be increased to reduce shear). The microfluidic flow channels can have various shapes to achieve a wide range of flow rates within the channels and to facilitate the integration and three-dimensional placement of biological tissues.

[0016] A "flow channel" is a path (such as linear, curved, single, multiple, network-like, etc.) through a medium (e.g., polymethyl methacrylate, etc.) that enables the movement of a fluid or a liquid or a gas. Thus, a flow channel can be connected to other components, i.e., the components can "communicate" with each other, and more specifically, "fluid communicate" with each other, and even more specifically, "liquid communicate" with each other. Such components include, but are not limited to, liquid inlet openings and ventilation holes. A "microfluidic flow channel" is a flow channel having dimensions greater than 1 micrometer and less than 1 millimeter.

[0017] According to the present invention, a "porous membrane" is a structure made of a biocompatible material and having pores of a size that allows only the exchange of gases and / or small chemical substances, or pores of a size that allows the movement and passage of large proteins, whole living cells, and / or parts thereof. Due to these properties, the porous membrane is also called a semipermeable membrane. The porous membrane may have porosity, flexibility, elasticity, flatness, or a combination thereof.

[0018] According to the present invention, the first microfluidic channel and the second microfluidic channel are "separated" by a porous membrane at least at their common portion. That is, at the common portion, the first surface of the porous membrane forms an interface with the lumen of the first microfluidic channel, and the second surface on the opposite side of the porous membrane forms an interface with the lumen of the second microfluidic channel. In other words, at this common portion, the first surface of the porous membrane serves as the boundary of the lumen of the first microfluidic channel, and the second surface of the porous membrane serves as the boundary of the lumen of the second microfluidic channel. Therefore, at this common portion, the first microfluidic channel and the second microfluidic channel run substantially parallel to each other adjacent to each other and are connected via the porous membrane. Outside this portion, the running paths of the first microfluidic channel and the second microfluidic channel may be different.

[0019] According to the present invention, the "capture structure" is a structure arranged at a specific position on the porous membrane, which can "fix" a biological substance, that is, a structure capable of attaching and adhering a biological substance in a predetermined manner for a long term or permanently. The capture structure is arranged at a "predetermined position" on or within the porous membrane, which means that the position of the capture structure is not irregularly distributed or the capture structure is not irregularly fabricated, but the capture structure is arranged by those skilled in the art at a clearly defined position in advance on or within the porous membrane.

[0020] The microfluidic device according to the present invention enables three-dimensional tissue culture in vitro in an advantageous manner by modeling the complex physiological microenvironment found in the human islet of Langerhans, i.e., the pancreas and its vicinity. This provides a biomimetic system (MPS) that allows biological materials, such as single cell spheroids (e.g., pseudo-islets), to self-guide to a determined position, be fixed in that position, and perform in situ analysis on the chip. This biological material is not separated by auxiliary structures as used in devices known in the art, but only by surrounding structures such as an extracellular matrix (ECM)-like hydrogel. Thus, the microfluidic device according to the present invention is a promising tool for supporting three-dimensional tissue culture of physiological models and the incorporation of related additional tissue components, and provides an in vitro platform for non-invasive analysis of cell (patho)physiology and responses to drug therapies.

[0021] In one embodiment of the present invention, the capture structure is a through-hole provided in a porous membrane, and the average diameter of the through-hole is preferably about 30 to 70 μm, more preferably about 40 to 60 μm, and even more preferably about 50 μm.

[0022] This means has the advantage that a structure in which the biological substance flowing through the first flow path can "stay" is provided in the porous membrane. In this case, the biological substance can be captured by the capture structure by appropriately adjusting the pressure conditions in the flow path. After the biological substance is introduced into the first microfluidic flow path, the pressure along the first microfluidic flow path is adjusted to be lower than the pressure applied to the porous membrane in the direction of the second microfluidic flow path within the first microfluidic flow path. Thereby, the biological substance stays in the hole-like structure and is fixed there. However, the dimensions can also be adjusted according to actual needs. For example, a large diameter of about 100 μm can be set to fix larger biological substances. Depending on the properties and materials of the porous membrane, through holes can be produced by methods known to those skilled in the art, for example, methods such as drilling, punching, cutting, etc., and in particular, can be produced using a UV laser cutter.

[0023] In another embodiment of the microfluidic device of the present invention, the average diameters of the first microfluidic flow path and the second microfluidic flow path are about 400 - 600 μm, and preferably about 500 μm.

[0024] This means has the advantage that it can meet the structural requirements for appropriately introducing biological substances such as cell spheroids with an average diameter of about 150 μm and liquids such as cell culture media into the flow path according to the principle of microfluidics. However, the dimensions can also be adjusted according to actual needs.

[0025] In still another embodiment of the microfluidic device of the present invention, the first microfluidic flow path has an inlet opening at the first end. In another embodiment, the first microfluidic flow path has a dead-end structure at the second end.

[0026] By this means, there is an advantage that biological substances that may be dissolved in a liquid and other components such as hydrogels can be introduced from the inlet opening into the microfluidic device or the first microfluidic channel. The dead-end structure may be disposed at the end of the channel provided in the support or carrier material surrounding the channel. By providing the dead-end structure, there is an advantage that the biological substances and other substances introduced into the first microfluidic channel can be retained in the channel to prevent outflow.

[0027] In another embodiment of the microfluidic device of the present invention, the second microfluidic channel has an inlet opening at a first end and an outlet opening at a second end.

[0028] By this means, for example, a fluid or liquid such as a culture medium for cell culture can be introduced from the inlet opening into the second microfluidic channel and discharged from the second microfluidic channel through the outlet opening. In this way, a fresh culture medium can be continuously supplied to the biological substance, particularly the biological substance fixed to the capture structure, through the fluid flow. Furthermore, a test substance such as a pharmacologically active substance can be introduced into the second channel, incubated for a desired adjustable time, rinsed, and discharged from the outlet opening.

[0029] In another embodiment of the microfluidic device of the present invention, the first microfluidic channel is provided on a first plate, and the second microfluidic channel is provided on a second plate.

[0030] The first plate and / or the second plate may be a carrier or support composed of polycarbonate glass, silicone, a material equivalent thereto, etc. The first microfluidic channel and / or the second microfluidic channel may be provided or incorporated on the first plate and / or the second plate. By providing the carrier plate, the microfluidic device can be managed and actual use becomes easy.

[0031] In yet another embodiment of the microfluidic device of the present invention, the first plate and / or the second plate is transparent.

[0032] By this means, it is possible to satisfy the structural requirements for observing and investigating the microfluidic device and the biological substances contained therein under a microscope, and in particular, for enabling the emitted light or fluorescence signal to be detected by an appropriate detector.

[0033] According to an embodiment of the present invention, the first plate and the second plate of the microfluidic device include materials selected from the group consisting of polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), thermoplastic elastomer (TPE), glass, cyclic olefin copolymer (COC), and the like.

[0034] This means has the advantage that it can ensure stability while being lightweight and can incorporate a microfluidic channel of a desired size into a material that can be designed to be transparent by a known and simple method.

[0035] In yet another embodiment of the microfluidic device of the present invention, the thickness of the porous membrane is about 10 to 30 μm, preferably about 20 to 25 μm, and more preferably about 22 μm.

[0036] According to the findings of the present inventors, the above dimensions are optimal for the porous membrane to achieve the object of the present invention and to be well attached to the microfluidic device.

[0037] In another embodiment of the microfluidic device according to the present invention, the porous membrane has pores, and the average diameter of the pores is about 1 to 10 μm, preferably about 1.5 to 8 μm, and more preferably about 2 to 3 μm.

[0038] According to the inventors, due to such pore diameters, semi-permeability is exhibited. Since it has such semi-permeability, only small molecules such as gases, metabolites, and active ingredients such as insulin can pass through the porous membrane, and biomaterials such as cells cannot pass through.

[0039] According to another embodiment of the microfluidic device of the present invention, the porous membrane contains polycarbonate (PC).

[0040] In this embodiment, a material that meets the requirements necessary for the proper functioning of the microfluidic device is used for the production of the porous membrane. However, alternative materials such as polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), vitrified hydrogel, electrospun polymer, etc. can also be used.

[0041] In another embodiment, the microfluidic device according to the present invention includes an upper plate adjacent to the second plate, and in another embodiment, includes a bottom plate adjacent to the first plate.

[0042] By providing the bottom plate and / or the upper plate, the stability required for active use in the laboratory is imparted to the microfluidic device. Also, holes can be provided in the upper plate to access or connect to the inlets and outlets of the first microfluidic channel and / or the second microfluidic channel.

[0043] In one embodiment of the present invention, the upper plate and / or the bottom plate may be configured to be transparent and may be made of a material selected from polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), thermoplastic elastomer (TPE), glass, cyclic olefin copolymer (COC), etc. In this regard, the foregoing description regarding the first plate and the second plate is equally applicable to the upper plate and the bottom plate.

[0044] In yet another embodiment of the microfluidic device of the present invention, the bottom plate is provided with a sensor.

[0045] Any type of non-contact sensor may be provided on the bottom plate. For example, an oxygen sensor can be provided. As the oxygen sensor, an oxygen indicator dye can also be used. The oxygen indicator dye can be disposed on the bottom plate.

[0046] In another embodiment of the microfluidic device of the present invention, the biological material preferably includes cell aggregates, preferably includes cell spheroids, more preferably includes cell spheroids containing insulin-producing cells, and even more preferably includes cell spheroids containing β-cells.

[0047] By this means, the microfluidic device according to the present invention can be further converted into a functional biosensor or a biomimetic system (MPS). A cell aggregate is a collection of living cells in the form of a functional complex. An example of a cell aggregate is a cell spheroid. A spheroid is a type of three-dimensional cell modeling that better mimics the environmental conditions of living cells compared to two-dimensional cell models. Specifically, it utilizes reactions between cells and reactions between the extracellular matrix (ECM) or ECM-like hydrogels and cells to mimic the environmental conditions of living cells. Usually, the average diameter of cell spheroids is about 120 to 300 μm. An example of a cell spheroid is a pseudo-islet. In such an embodiment, a biomimetic pancreatic chip platform is provided. Therefore, in another embodiment of the present invention, the biological material preferably includes insulin-producing cells and preferably includes β-cells.

[0048] Another object of the present invention relates to a microfluidic system comprising the microfluidic device according to the present invention and a fluid source in fluid communication with a second flow path.

[0049] The features, characteristics, and advantages described with respect to the microfluidic device of the present invention are equally applicable to the microfluidic system according to the present invention.

[0050] Another object of the present invention is a method for culturing a biological substance, comprising: a) providing a microfluidic device according to the present invention; b) introducing the biological substance into a first microfluidic channel; c) introducing a culture medium into a second microfluidic channel; and d) culturing the biological substance under conditions where the biological substance functions physiologically. The features, characteristics and advantages described for the microfluidic device of the present invention are equally applicable to the method according to the present invention.

[0051] In one embodiment of the method according to the present invention, in such a context, the biological substance comprises cell aggregates, preferably cell spheroids, preferably cell spheroids comprising insulin-producing cells, and more preferably cell spheroids comprising β-cells.

[0052] In another embodiment of the method according to the present invention, in step (b), a further substance is introduced into the first microfluidic channel. Preferably, this further substance comprises living cells and / or a hydrogel, preferably an extracellular matrix (ECM)-like hydrogel.

[0053] By this means, an environment almost identical to the natural environment can be advantageously constructed for the biological substance or cell spheroid. Since the interaction between living cells and / or the interaction between living cells and ECM becomes possible, in particular, it is possible to simulate and investigate the function of the pancreas.

[0054] In another embodiment of the method of the present invention, in step (b), the introduction of the biological substance into the first microfluidic channel is achieved by a flow generated by a hydrostatic pressure.

[0055]

[0056] ​By this means, a physical force can be applied to the microfluidic device in an advantageous manner to transport the biological substance to a desired position. In this embodiment, the pressure along the first microfluidic channel is adjusted such that it is lower than the pressure applied to the porous membrane in the direction of the second microfluidic channel within the first microfluidic channel. Thereby, the biological substance remains in the hole-shaped capture structure and is fixed therein. This adjustment can be performed by providing an appropriate height difference between the inlet opening of the first microchannel and the outlet opening of the second microchannel.

[0057] In another embodiment of the method according to the invention, the method according to the invention e’) investigating the biological substance, preferably visually, more preferably by microscopy and / or spectroscopic analysis further comprises.

[0058] In yet another embodiment, the method according to the invention c’) discharging the medium from the second microfluidic channel, preferably investigating the discharged medium, more preferably investigating the discharged medium for compounds secreted by the biological substance further comprises.

[0059] The foregoing features and the features described hereinafter cannot be used only in the combinations shown in each embodiment, but can also be used in other combinations or alone without departing from the scope of the present invention.

Brief Description of the Drawings

[0060] The present invention will be described and explained in more detail with reference to the following examples and drawings, but the present invention is not limited to these examples and drawings.

Figure 1

Figure 2

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Figure 5

Example

[0061] 1. Summary The inventors disclose a biomimetic system (MPS) incorporating a microfluidic device for the purpose of modeling a complex physiological microenvironment in vitro. The inventors, as an example, constructed an in vivo-like environment of pancreatic islets. The constructed pancreatic endocrine gland chip is a special microfluidic system that can self-align and fix individual spheroids in a predetermined position, enabling in situ analysis on the chip. Pancreatic β-cells are aggregated into a three-dimensional cell cluster called "pseudo-islets" and introduced into the microfluidic system. Furthermore, by embedding the pseudo-islets in an ECM-like hydrogel that mimics the physiological microenvironment, it can be integrated with the tissue-related types of cells, thereby improving the culture conditions of the biomimetic pancreatic chip platform.

[0062] Since optical access to the tissue on the chip is easy, non-invasive and real-time imaging can be performed, thereby enabling the monitoring of survival rate and functionality. To temporally track the insulin secretion dynamics and metabolic activities in response to glucose stimulation on the chip, dynamic sampling of the effluent and online non-invasive real-time monitoring of oxygen consumption using an integrated optical oxygen sensor were employed. Treatment with glucose increased insulin secretion and oxygen consumption, confirming the on-chip cell functionality and enhanced metabolic activity of glucose-stimulated islets.

[0063] In vitro studies of the pancreas are very important for elucidating mechanisms, pharmaceutical research, and therapeutic advancements in the field of diabetes (DM). The biomimetic system (MPS) disclosed herein is a promising tool that supports, for example, three-dimensional tissue culture such as islet modeling and the incorporation of additional relevant tissue components, and provides an in vitro platform for non-invasive analysis of cell (patho)physiology and responses to drug treatment.

[0064] 2. Materials and Methods Design and Fabrication of Microfluidic Devices In FIG. 1A, a disassembled schematic view of an embodiment of the microfluidic device according to the present invention is indicated by reference numeral 10. The microfluidic device of the present invention has a sandwich structure. The microfluidic device of the present invention includes a first microfluidic channel 12, a second microfluidic channel 14, and a porous membrane 16. The first microfluidic channel 12 is installed or embedded in a first plate 26, and the second microfluidic channel 14 is embedded in a second plate 28. In this regard, the first plate 26 and the second plate 28 are overlapped with the porous membrane 16 sandwiched therebetween and are aligned with the porous membrane 16, so that the first microfluidic channel 12 and the second microfluidic channel 14 are substantially parallel to each other at portion s and are separated only by the porous membrane 16.

[0065] The first microfluidic channel 12 has an inlet opening 19 at its first end and a dead-end structure 20 at its second end. The second microfluidic channel 14 has an inlet opening 22 at its first end and an outlet opening 24 at its second end, which is behind or downstream of the common portion s.

[0066] The first plate 26 has a bottom plate 30 adjacent to its bottom surface, and the second plate 28 has an upper plate 32 adjacent to its upper surface. The porous membrane 16, the second plate 28, and the upper plate 32 each have through-holes 38, 40, 42 aligned with the inlet opening 19 of the first microfluidic channel 12. Further, the upper plate 32 further has an opening 34 in the form of a through-hole aligned with the inlet opening 22 of the second plate 28. Furthermore, the upper plate 32 further has another opening 36 in the form of a through-hole aligned with the outlet opening 24 of the second plate 28.

[0067] As shown in the enlarged view in FIG. 1B, the porous membrane 16 has so-called capture structures, three of which are indicated by reference numeral 18. The capture structures are formed as through-holes.

[0068] The microfluidic device 10 according to the present invention is also called a "chip" or a "biomimetic chip". All of the chip designs were performed using design software with a computer.

[0069] The inventors used PMMA (Plexiglas Resist, Evonik), PC (ipCELLCULTURE TMUsing various materials including a track-etched membrane (it4ip) and PDMS (Sylgard 184, Dow Corning), a microfluidic device 10, i.e., a chip, was fabricated as a prototype. In its simplest configuration, the microfluidic device 10, i.e., the chip, comprises a PMMA medium layer ( = second plate 28) with a thickness of 250 μm and a PMMA tissue layer ( = first plate 26). These layers have flow channels and chambers of a specific shape and are separated from each other by a thin porous PC membrane ( = porous membrane 16) with a thickness of about 22 μm (Figure 1B). The medium flow channel ( = second microfluidic flow channel 14) and the tissue chamber ( = first microfluidic flow channel 12) both feature a width of about 500 μm, and the width of the tissue flow channel ( = first microfluidic flow channel 12) connected to this tissue chamber is about 250 μm. The PMMA upper layer ( = second plate 28) with a thickness of about 250 μm is covered by a flexible PDMS slab ( = upper plate 32) with a thickness of about 3 mm. This slab serves as an interface for tube connection and enables access to the inlets ( = inlet openings 19; through-holes 38, 40, 42) and outlets ( = outlet openings 24; opening 36) of the microfluidic flow channel structure ( = first microfluidic flow channel 12 and second microfluidic flow channel 14). The bottom layer ( = bottom plate 30) is as thin as about 175 μm, enabling optical access and, when a sensor is incorporated into this microfluidic system, also functioning as a sensor substrate. In this case, an oxygen indicator dye is linearly arranged with a width of about 250 μm at the position of the tissue chamber on the bottom layer ( = bottom plate 30). Any type of non-contact optical sensor can be incorporated into the microfluidic system using any of the layers included in the chip as a sensor substrate.

[0070] The PMMA layers ( = first plate 26, second plate 28) are CO 2The PC membrane (=porous membrane 16) was formed using a laser cutter, specifically a UV laser cutter. The capture structure (18) with a specific shape on the PC membrane (=porous membrane 16) was fabricated by opening holes of approximately 50 μm using a laser cutter. To prepare the PDMS slabs (=bottom plate 30; upper plate 32) used as the connection layer, a PDMS prepolymer and a curing agent were mixed at a weight ratio of 10:1. To mold the slabs, 40 g of the uncured mixture was poured into a square Petri dish and cured overnight at 60 °C. Next, the obtained PDMS was pre-scored into the shape of the chip using a laser cutter and then cut out with a scalpel. The inlet and outlet were perforated with a biopsy punch of 0.35 mm or 0.75 mm. The fabricated PDMS connection layer was functionalized with APTES and bonded to the PMMA upper layer by activating it with O 2 plasma.

[0071] First, the positions of the two PMMA layers (=first plate 26, second plate 28) were aligned, and these two PMMA layers (=first plate 26, second plate 28) were sandwiched between two microscope slides, and pressure was applied from both sides with double clips and heated at 125 - 130 °C in a preheated air-circulating thermostatic oven (Memmert) for bonding. The assembly of the chip (=microfluidic device 10) was carried out by the following two consecutive bonding steps. (i) In the first step, the PC membrane (=porous membrane 16) was bonded to the culture medium flow path (the second microfluidic flow path 14), and (ii) in the second step, the entire chip (=microfluidic device 10) was assembled. Each step was performed for 15 minutes.

[0072] 2 In the glucose-stimulated insulin secretion (GSIS) experiment, the fabricated PDMS well (h = 3 mm, φ = 4 mm) was placed above the outlet (=opening 36) of the culture medium flow path (=the second microfluidic flow path 14) and bonded by plasma activation.

[0073] Cell Culture and Formation of Pseudoislets ​The pancreatic β-cell line INS-1E derived from rat insulinoma was used in the experiment. In a T25 cell culture flask (seeding density: 40,000 cells / cm 2 ), INS-1E cells were cultured under standard conditions (37 °C, 5% CO 2 , 20% O 2 , 95% humidity) and passaged at 70 - 80% confluence using a 0.05% trypsin / EDTA solution (Gibco). The culture medium RPMI 1640 was supplemented with 10 mM Hepes buffer solution (Gibco), 1 mM sodium pyruvate (Gibco), 50 μM β-mercaptoethanol (Gibco), 5% FCS (HyClone Fetal Clone II, GE Life Sciences), and 1% penicillin-streptomycin (stock solution: 10,000 U / mL penicillin, 10 mg / mL streptomycin; Gibco).

[0074] To form pseudo-islets, 100 μL of medium was placed in each well of a 96-well ultra-low attachment surface (ULA) round-bottom plate (Greiner Bio-one) and INS-1E cells were seeded at a concentration of 500 cells / well according to a method reported in the art. Pseudo-islets were formed by culturing for 72 hours under standard cell culture conditions and then injected into the chip system.

[0075] Introduction of Tissues and Hydrogels into the Chip System and On-Chip Culture Before injecting the cells, the chip was treated with oxygen plasma for 5 minutes to hydrophilize it. Next, with the pipette tip at the tissue chamber inlet and the inlets and outlets of the medium flow path open, the chip was flushed with 70% ethanol and then washed three times with PBS.

[0076] Sphéroïdes ont été rassemblés dans un puits de la plaque ULA et injectés dans la pipette à l'entrée de la chambre tissulaire. Ensuite, le hydrogel (FibriCol, Advanced Biomatrix) a été introduit dans la puce microfluidique à partir de la pipette placée à l'entrée de la voie tissulaire. Dans ce cas, d'autres types de cellules incluses dans l'hydrogel peuvent être introduites ensemble. Ensuite, la puce a été laissée en repos dans des conditions de culture cellulaire standard pour faire réticuler l'hydrogel. Après incubation pendant 60 minutes, la pipette à l'entrée de la voie tissulaire a été retirée et la chambre tissulaire a été scellée hermétiquement avec un film adhésif pour PCR. Ensuite, un tube en silicone (VERNAAAD04103, VWR international) a été utilisé pour connecter la voie d'écoulement de milieu de la puce à un dispositif de pompe à seringue à 12 canaux (Landgraf Laborsysteme HLL), et une pression positive a été appliquée pour effectuer une perfusion à 20 μL / h. 2 La puce a été cultivée dans un incubateur à 5 % de CO

[0077] Cell Viability Staining La coloration des cellules vivantes / mortes a été effectuée sur la puce en visualisant les cellules vivantes avec de la fluorescéine diacétate (FDA, Thermo Fisher) à 27 μg / mL et en marquant les cellules mortes avec de la propidium iodure (PI, Sigma-Aldrich) à 135 μg / mL. Les noyaux cellulaires ont été colorés avec de la DAPI (Thermo Fisher) à 1 μg / mL. Le jour de l'évaluation du taux de survie, la puce a été retirée du dispositif de perfusion et lavée avec du PBS+ en utilisant le flux de gravité. Ensuite, une solution de coloration contenant FDA, PI et DAPI dans le PBS a été préparée et injectée dans la voie d'écoulement de milieu du système de puce en utilisant le flux de gravité, puis incubée pendant 15 minutes. La puce a été lavée 3 fois avec du PBS- et photographiée immédiatement au microscope confocal (LSM 710 de Zeiss et microscope à disque tournant Axio Z.1 Cell Observer de Zeiss).

[0078] Immunofluorescence Staining Pour étudier l'intégrité, la structure et la fonction des tissus sur la puce, des colorations pour visualiser l'E-cadhérine, l'insuline, l'actine F et la DAPI ont été effectuées.

[0079] All washing and staining solutions were flushed into the medium flow path using gravity flow. Before fixing the cells, the chip was removed from the pump device and flushed with PBS+. The cells were fixed by incubating with 4% Roti® Histofix (Carl Roth) for 20 minutes. After thorough washing with PBS-, the chip was stored at 4 °C until the next treatment. Non-specific binding was inhibited and the cells were made transparent by incubating with a PBS- solution containing 0.1% Triton-X100 and 3% normal donkey serum for 1 hour. The anti-insulin antibody (1:200, ab181547, abcam) and anti-E-cadherin antibody (1:50, BD610181, BD Biosciences) as primary antibodies were diluted with an antibody diluent (PBS- solution containing 0.01% Trition-X100 and 0.3% normal donkey serum), incubated at room temperature for 2 hours, and then incubated at 4 °C overnight. After thoroughly washing the chip by flushing the medium flow path 3 times with a washing buffer (PBS- solution containing 0.01% Triton-X100 and 0.3% normal donkey serum), it was incubated with the labeled secondary antibodies and DAPI. The Alexa Fluor 647 donkey anti-rabbit antibody (1:100, A31573, Thermo Fisher), Alexa Fluor 488 donkey anti-mouse antibody (1:100, A21202, Thermo Fisher), Alexa Fluor 546 phalloidin (1:100, A22283, Thermo Fisher), and DAPI (1:1000, MBD0015, Merck) as secondary antibodies were diluted with the antibody diluent, incubated at room temperature for 2 hours, and then thoroughly washed 3 times with the washing buffer. After storing the chip in PBS- at 4 °C, images were taken with an Axio Z.1 Cell Observer spinning disk type confocal microscope (Carl Zeiss).

[0080] Glucose-Stimulated Insulin Secretion Assay (GSIS) on Chip As described above, cells were introduced into the chip and cultured overnight (20 μL / h). The chip was connected to a 4-port microfluidic valve so that switching between different conditions could be performed. Before perfusion under low glucose conditions (3.3 mM in KREBS buffer) and high glucose conditions (16.7 mM in KREBS buffer), the chip was perfused with 1×KREBS buffer containing 25 mM Hepes (Gibco), 0.1% BSA (Sigma-Aldrich), and 0 mM glucose for 1 hour to perform a cell synchronization step. Next, the chip was perfused for 1 hour each under low glucose conditions, high glucose conditions, and low glucose conditions. The effluent was collected every 60 minutes from the well above the outlet of the medium flow path and stored at -20 °C until insulin analysis was performed. The sampling time was calculated based on the volumes of the chip, tube, and valve. Insulin secretion was quantified using a rat insulin ELISA kit (Mercodia) according to the manufacturer's instructions.

[0081] 3. Results Concept and Design of Microfluidic Chip To construct a biomimetic platform for modeling the physiology and pathology of pancreatic endocrine glands, several important features of the in vivo microenvironment were considered. In this specification, insulin-secreting β-cells form part of a three-dimensional cell cluster, which is embedded in a unique microenvironment with extensive angiogenesis. To achieve this, the microfluidic platform was configured as a custom-made multilayer hybrid device featuring at least two channel geometries separated by a semipermeable membrane (Figure 1A). This microfluidic chip is mainly fabricated using PMMA, which has less absorption of hydrophobic molecules compared to PDMS, a material commonly used in the art. By directly incorporating a capture structure into the semipermeable membrane (Figure 1B), cell spheroids can be fixed at a predetermined position in the lower channel of the microfluidic chip, enabling analysis by time-resolved readout methods (illustrated in Figure 1C). Since spheroid introduction enables self-guided capture, spheroids can be incorporated into the microfluidic system (Figure 1D).

[0082] In vitro culture of tissues under physiological conditions after the introduction of spheroids can be carried out by this chip device, and furthermore, an extracellular matrix (ECM)-like hydrogel can be incorporated and co-cultured with another type of cell related to the tissue. The culture medium is supplied by the flow of microscale dynamic fluid in the upper flow path above the lower flow path. By controlling the fluid flow, not only can nutrients be stably supplied and waste products be removed, but also the effluent can be dynamically collected to investigate the secretion kinetics such as glucose-stimulated insulin secretion, which is an important function of β cells, in a time-resolved manner. Furthermore, since a non-contact optical sensor such as a non-contact optical sensor for oxygen measurement can be incorporated into the microfluidic system, the oxygen consumption by cells can be monitored non-invasively and in real time directly on the chip online. The shape of the microfluidic system can be adjusted according to spheroids of various sizes and numbers. Depending on the purpose and application of the microfluidic system, the microfluidic system can be manufactured using various materials and thus can be variously improved and configured, so various types of port connections are possible for controlling the fluid flow on the chip.

[0083] Integration of Tissues into the Chip System To develop the chip, an in vitro model established in the past that mimics the insulin-secreting endocrine function of β cells was used. By culturing for 72 hours at a density of 500 cells / well using an ultra-low adhesion surface U-bottom well plate, the cells were spontaneously aggregated to a mean diameter of up to 150 μm to form pseudo-islets showing important physiological cell-cell contacts.

[0084] According to the injection protocol developed in the present invention that utilizes the flow generated by the hydrostatic pressure due to the height difference between the inlet for the tissue and the outlet of the culture medium flow path, the desired number of spheroids and extracellular matrix-like hydrogels corresponding to the number of captures on the chip can be introduced into the microfluidic system (Figure 1D). Using this introduction principle, the spheroids are guided to predetermined positions on the chip. The hydrogel holds the three-dimensional β-cell-like microtissue in place during on-chip culture and mimics the in vivo microenvironment of these cells. Since another type of cell can be easily incorporated into the hydrogel, additional components related to the tissue (e.g., endothelial cells) can be incorporated. To confirm that the hydrogel was uniformly introduced into the flow path, fluorescent microspheres were incorporated into the hydrogel before injecting the hydrogel into the chip (Figure 1E). After culturing the chip system overnight and obtaining the maximum intensity projection images of the z-stack, it was confirmed that the hydrogel was uniformly filled throughout the tissue flow path.

[0085] Evaluation of Viability on Chip To examine the effect of the introduction operation onto the chip and on-chip culture on the viability of the tissue on the chip, live / dead cell staining using FDA / PI was performed, and it was shown that most of the cells on the chip were viable and a few dead cells were scattered among the aggregates (Figure 2).

[0086] Evaluation of Structure and Functionality of Tissues on Chip Expression of Insulin and E-Cadherin on Chip To evaluate the integrity and functionality of the pseudo-islets on the chip, the expression of E-cadherin and insulin was analyzed. E-cadherin is important as an intercellular adhesion protein and also affects the insulin-secreting ability of β cells. From the obtained confocal microscope images, it was shown that E-cadherin and insulin maintained high expression in the pseudo-islets on the chip and that the cells as a whole exhibited their original insulin production (Figure 3).

[0087] Glucose-Stimulated Insulin Secretion on Chip An important function of β cells is to secrete insulin in response to glucose. To analyze the functionality on the chip, cells were continuously incubated under low glucose concentration conditions and high glucose concentration conditions to perform a glucose-stimulated insulin secretion (GSIS) assay. The β cells on the chip showed glucose responsiveness in that insulin secretion increased in response to high glucose stimulation and decreased upon exposure to low glucose (Figure 4).

[0088] On-Chip Co-Culture Using Tissue-Related Types of Cells In vivo, angiogenesis is actively occurring in pancreatic islets, and there is a strong functional and physical interaction between β cells and endothelial cells. Furthermore, endothelial cells assist in glucose sensing and endocrine hormone secretion, and the importance of the proximity of such cell types has been emphasized. The microfluidic chip device of the present invention can incorporate another type of cell embedded in a hydrogel into a tissue chamber. Based on this, endothelial cells were suspended in a mixture of type I collagen and fibrin and introduced into a chip system containing pseudo-pancreatic islets. When visualized with CellTracker in the hydrogel, the success of cell introduction and the three-dimensional distribution of the introduced cells could be confirmed (Figure 5A). During a 2-day culture period, immunofluorescent staining of CD31 was performed, and it was shown that the cells on the chip had already started to aggregate and form a three-dimensional vascular-like structure. When the expression of endogenous insulin was confirmed, it was shown that the pseudo-pancreatic islets retained high functionality (Figure 5B).

Claims

1. A microfluidic device (10) comprising: a first microfluidic channel (12), a second microfluidic channel (14), and a porous membrane (16); at least a portion (s) of the first microfluidic channel (12) and the second microfluidic channel (14) being separated by the porous membrane (16); the porous membrane (16) having a capture structure (18) configured to immobilize a biological substance at a predetermined position; a microfluidic device.

2. The microfluidic device (10) according to claim 1, wherein the capture structure (18) is a through-hole provided in the porous membrane (16).

3. The microfluidic device (10) according to claim 2, wherein the average diameter of the through-hole is about 30 to 70 μm, preferably about 40 to 60 μm, and more preferably about 50 μm.

4. The microfluidic device (10) according to any one of the preceding claims, wherein the average diameters of the first microfluidic channel (12) and the second microfluidic channel (14) are about 400 to 600 μm, preferably about 500 μm.

5. The microfluidic device (10) according to any one of the preceding claims, wherein the first microfluidic channel (12) has an inlet opening (19) at a first end.

6. The microfluidic device (10) according to any one of the preceding claims, wherein the first microfluidic channel (12) has a dead-end structure (20) at a second end.

7. The microfluidic device (10) according to any one of the preceding claims, wherein the second microfluidic channel (14) has an inlet opening (22) at a first end and / or an outlet opening (24) at a second end.

8. The microfluidic device (10) according to any one of the preceding claims, wherein the first microfluidic channel (12) is provided in a first plate (26) and / or the second microfluidic channel (12) is provided in a second plate (28).

9. The microfluidic device (10) according to claim 8, wherein the first plate (26) and / or the second plate (28) is transparent.

10. The microfluidic device (10) according to claim 8 or 9, wherein the first plate (26) and the second plate (28) comprise a material selected from the group consisting of polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), thermoplastic elastomer (TPE), glass, and cyclic olefin copolymer (COC).

11. The microfluidic device (10) according to any one of the preceding claims, wherein the thickness of the porous membrane (16) is about 10 to 30 μm, preferably about 20 to 25 μm, and more preferably about 22 μm.

12. The microfluidic device (10) according to any one of the preceding claims, wherein the porous membrane (16) has pores with an average diameter of about 1 to 10 μm, preferably about 1.5 to 8 μm, and more preferably about 2 to 3 μm.

13. The microfluidic device (10) according to any one of the preceding claims, wherein the porous membrane comprises polycarbonate (PC).

14. The microfluidic device (10) according to any one of claims 8 to 13, comprising a bottom plate (30) adjacent to the first plate (26).

15. The microfluidic device (10) according to any one of claims 8 to 14, comprising an upper plate (32) adjacent to the second plate (28).

16. The microfluidic device (10) according to claim 15, wherein the upper plate (32) has openings (34, 36) connecting to the inlet opening (22) and / or the outlet opening (24).

17. The microfluidic device (10) according to any one of claims 14 to 16, wherein the bottom plate (30) and / or the upper plate (32) is transparent.

18. The microfluidic device (10) according to any one of claims 14 to 17, wherein the bottom plate (30) and / or the upper plate (32) comprise a material selected from the group consisting of polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), thermoplastic elastomer (TPE), glass, and cyclic olefin copolymer (COC).

19. The microfluidic device (10) according to any one of claims 16 to 18, wherein the bottom plate (30) comprises a sensor, preferably an oxygen sensor.

20. The microfluidic device (10) according to any one of the preceding claims, wherein the biological material includes cell aggregates, preferably includes cell spheroids, more preferably includes cell spheroids containing insulin-producing cells, and even more preferably includes cell spheroids containing β cells.

21. A microfluidic system comprising the microfluidic device (10) according to any one of claims 1 to 20 and a fluid source in fluid communication with a second flow path (12).

22. A method for culturing a biological material, comprising: a) providing a microfluidic device according to any one of claims 1 to 20; b) introducing the biological material into a first microfluidic flow path; c) introducing a culture medium into a second microfluidic flow path; and d) culturing the biological material under conditions in which the biological material functions physiologically. A method including these steps.

23. The method according to claim 22, wherein the biological material includes cell aggregates, preferably includes cell spheroids, more preferably includes cell spheroids containing insulin-producing cells, and even more preferably includes cell spheroids containing β cells.

24. The method according to any one of the preceding claims, wherein in step (b), a further substance is introduced into the first microfluidic flow path.

25. The method according to claim 24, wherein the further substance includes living cells and / or a hydrogel, preferably an extracellular matrix (ECM)-like hydrogel.

26. The method according to any one of the preceding claims, wherein the introduction in step (b) is achieved by a flow generated by hydrostatic pressure.

27. e') further comprising the step of examining the biological material, preferably visually, more preferably by microscopic observation and / or spectroscopic analysis. The method according to any one of the preceding claims.

28. The method according to any one of the preceding claims, further comprising the step of discharging the culture medium from the second microfluidic flow path.

29. The method according to claim 28, wherein the discharged culture medium is examined, preferably for compounds secreted from the biological material.

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