Chip, microfluidic cell culture device including the same, and method for cell culture using the same

Abstract

A wafer includes a first substrate, a second substrate coupled to the first substrate, and a membrane disposed between the first and second substrates. The first substrate has a first channel, a first trench, a second trench, and a third trench, and the second substrate has a second channel corresponding to the first channel. The first trench, the second trench, and the third trench extend from the first channel, and each trench forms an opening at the edge of the first substrate. A microfluidic cell culture device includes a wafer, an incubator, and a potentiostat. A method of culturing cells includes providing the microfluidic cell culture device and introducing cells into a membrane.

Description

[Technical Field]

[0001] This disclosure relates to a chip and a microfluidic cell culture device comprising the chip, and more particularly to a chip for providing a cell culture environment with a gas-liquid interface and simulating cell microphysiological functions in vitro and a microfluidic cell culture device comprising the chip. [Previous Technology]

[0002] An organ-on-chip (OoC) is a system that contains engineered / natural micro-tissues or specific organs cultured within a wafer with a microfluidic structure and provides a cell culture environment for simulating the microphysiological functions of engineered / natural micro-tissues or specific organs in vitro.

[0003] OoC (Out-of-Cell) technology holds promise as an alternative to animal testing, potentially advancing the discovery and development of advanced drugs. The air-liquid interface (ALI) of the wafer creates an environment where air and liquid layers coexist, which is crucial for cell culture. For example, lung epithelial cells grown in wafers with an ALI can differentiate into a periciliary liquid layer (PCL) and secrete a mucus layer (ML), thus reproducing mucociliary clearance (MCC). Furthermore, previous studies have shown that lung epithelial cells in wafers with an ALI express higher levels of tight junctions (acting as an epithelial barrier to prevent harmful or infectious substances from entering the body) than those in conventional cell culture environments.

[0004] In a wafer with an air-liquid interface, the natural transport, precipitation and permeation patterns of microparticles (such as particulate matter and aerosols) in the air between lung tissues can be maintained, and the concentration of microparticles can be prevented from being diluted or affected by the culture medium in the wafer.

[0005] Traditionally, OoC analysis relies on invasive endpoint analysis, such as large, sophisticated instruments, expensive antibodies, and cumbersome labeling. It lacks essential features such as in situ, miniaturization, real-time measurement, and long-term efficacy, and cannot continuously observe cellular changes. Therefore, there is a pressing need in the art to provide a long-term, non-invasive, label-free, real-time, and time-varying sensing method for analyzing changes in cell proliferation, tight junction formation, ciliary differentiation, mucus secretion, cell layer permeability, or cell density grown in an OoC system.

[0006] The above requirements can be overcome by using electrical signal measurement. Specifically, electrodes are placed in the microfluidic channels at the top and bottom of the OoC system. In the conventional transepithelial electric resistance (TEER) measurement method, the resistance value is obtained by applying current through the cell; however, the air layer at the gas-liquid interface blocks the current (because current transmission in electrode sensing requires a substrate), and the electrodes cannot detect the internal cells.

[0007] In recent years, electrochemical cell impedance spectroscopy (ECIS) has been proposed, aiming to avoid the influence of air by having the cells in direct contact with the electrodes. However, most studies using ECIS to detect cells involve immersing the electrodes in the cell culture medium or temporarily interrupting the ALI state during the sensing process.

[0008] In view of this, there is still an urgent need in the art to provide a novel microfluidic cell culture device with an OoC system that can perform electrochemical impedance spectroscopy (EIS) sensing in an ALI environment, so as to overcome the shortcomings of the prior art. [Summary of the Invention]

[0009] Other aspects of this disclosure will be set forth in the following description, and will become apparent to those skilled in the art upon careful reading of the following. Those skilled in the art will also be able to deduce the contents of this disclosure from the embodiments thereof. The advantages of this disclosure can be understood and realized from the portions specifically pointed out in the appended claims.

[0010] To address the aforementioned problems, this disclosure provides a wafer comprising: a first substrate; a second substrate coupled to the first substrate; and a film disposed between the first substrate and the second substrate. The first substrate may include: a first channel; a first trench extending from the first channel and having a first opening formed on an edge of the first substrate; a second trench extending from the first channel and having a second opening formed on an edge of the first substrate; and a third trench extending from the first channel and having a third opening formed on an edge of the first substrate. The second substrate may include a second channel corresponding to the first channel.

[0011] This disclosure further provides a microfluidic cell culture device, comprising: a wafer; an incubator housing the wafer to provide a culture medium flow in a second channel; and a potentiostat coupled to a first electrode, a second electrode, and a third electrode to record the electrochemical impedance spectrum of cells. The wafer may comprise: a first substrate; a second substrate coupled to the first substrate; and a membrane disposed between the first substrate and the second substrate. The first substrate may comprise: a first channel; a first trench extending from the first channel and having a first opening formed on an edge of the first substrate; a second trench extending from the first channel and having a second opening formed on an edge of the first substrate; a third trench extending from the first channel and having a third opening formed on an edge of the first substrate; a first electrode partially disposed in the first trench; a second electrode partially disposed in the second trench; and a third electrode partially disposed in the third trench. The second substrate may comprise a second channel corresponding to the first channel.

[0012] This disclosure also provides a method for culturing cells, comprising: providing a microfluidic cell culture device; and introducing cells into a membrane.

Implementation Method

[0014] Cross-reference to related applications

[0015] This application claims priority to U.S. Provisional Application No. 63 / 644,517, filed on May 9, 2024, the entire contents of which are incorporated herein by reference.

[0016] The following description of embodiments illustrates the implementation of this disclosure. Those skilled in the art to which this disclosure pertains can easily understand the advantages and effects of this disclosure based on the content contained herein. However, the embodiments described in this disclosure are not intended to limit the scope of this disclosure. This disclosure can also be implemented or applied by other different implementation methods, and the details described in this disclosure can also be given different changes or modifications according to different viewpoints and applications, without departing from the spirit of this disclosure.

[0017] The proportions, structures, sizes, and other features shown in the accompanying drawings of this disclosure are only for the purpose of assisting those skilled in the art in reading and understanding this disclosure, and are not intended to limit the scope of its implementation. Therefore, any changes in proportions, modifications to the structure, or adjustments to the size, without affecting the purpose and effects of this disclosure, shall fall within the scope of the technical content disclosed herein.

[0018] As used herein, when “comprising,” “including,” or “having” a particular element, it may include other elements, structures, areas, parts, devices, systems, or connections, etc., unless otherwise stated, rather than excluding such other elements.

[0019] As used herein, terms such as "on," "above," "below," "top," "side," "front," "between," and "in the middle" are used only for the purpose of illustrating the embodiments disclosed herein and are not intended to limit the scope of this disclosure. Any adjustment, interchange, or alteration of relative positions and relationships, without substantially changing the technical content of this disclosure, shall fall within the protection scope of this disclosure.

[0020] As used herein, the terms "first," "second," and "third" are used only for the convenience of describing or distinguishing elements, structures, regions, parts, devices, or systems, and are not intended to limit the scope of this disclosure or to limit the spatial order of such elements. Furthermore, unless otherwise expressly stated herein, the singular forms "a" and "the" used herein also include the plural forms, and the word "or" used herein is interchangeable with "and / or."

[0021] Unless otherwise expressly stated herein, the terms “layer” and “phase” used herein are used interchangeably.

[0022] In some embodiments of this disclosure, the first electrode, the second electrode and the third electrode are inserted into one side of the wafer disclosed herein for use in detecting cells cultured in the wafer disclosed herein by electrochemical cell impedance spectroscopy (ECIS) in a gas-liquid interface environment.

[0023] In some embodiments of this disclosure, the microfluidic cell culture device can distinguish different tissue layers, such as the mucus layer, ciliary layer, and cell layer, in a non-invasive and label-free manner. In some embodiments of this disclosure, the gas-liquid interface of the microfluidic cell culture device can introduce atomized drugs for real-time monitoring of different tissue layers.

[0024] In some embodiments of this disclosure, since the electrode-electrolyte interface is a series of continuous processes occurring at different time points (e.g., mass transfer and charge transfer processes), electrochemical impedance spectroscopy (EIS) is needed to assist in analyzing time-dependent mechanisms when analyzing cell-related electrical signal changes. In some embodiments of this disclosure, the impedance of the electrochemical system is calculated by applying alternating current (AC) potentials of different frequencies to the electrochemical system and measuring the AC signal. In some embodiments of this disclosure, the electrochemical reactions at the electrode interface of the system are represented by an equivalent circuit composed of circuit elements.

[0025] In some embodiments of this disclosure, the microfluidic cell culture device of this disclosure provides non-invasive impedance sensing cell technology in a two-phase environment (i.e., an environment with a gas-liquid interface).

[0026] In some embodiments of this disclosure, the microfluidic cell culture device disclosed herein is suitable for any cell tissue that requires a gas-liquid interface environment.

[0027] This relatively simple process overcomes the bottleneck of previous cell electrical impedance measurements at the gas-liquid interface, while maintaining a stable culture environment in the microfluidic channel without damaging the cells. Through electrical signal analysis, endpoint analysis methods that previously required hours or even more than a day can be shortened to minutes, ultimately achieving non-invasive, immediate, and long-term repeatable measurements.

[0028] In at least one embodiment of this disclosure, the first channel may be coated with an extracellular matrix.

[0029] In at least one embodiment of this disclosure, the first substrate and / or the second substrate may comprise polycarbonate and / or polydimethylsiloxane.

[0030] In at least one embodiment of this disclosure, the wafer may include: a first electrode, partially disposed on a first trench; a second electrode, partially disposed on a second trench; and a third electrode, partially disposed on a third trench.

[0031] In at least one embodiment of this disclosure, at least a portion of the first electrode may be disposed on the first trench through the first opening; at least a portion of the second electrode may be disposed on the second trench through the second opening; and at least a portion of the third electrode may be disposed on the third trench through the third opening.

[0032] In at least one embodiment of this disclosure, the membrane may be porous and contain polyethylene terephthalate.

[0033] In at least one embodiment of this disclosure, the wafer may further include a first culture medium disposed on a first channel and / or a second channel. In some embodiments of this disclosure, the first culture medium may be selected from the group consisting of EX plus medium, respiratory epithelial cell basal medium, respiratory epithelial cell growth medium, bronchial epithelial cell growth medium, F12K medium, Eagle's minimum essential medium, Dulbecco's modified Eagle's medium, and any combination thereof. In some embodiments of this disclosure, the second culture medium may be selected from the group consisting of PneumaCult™-ALI medium, StemCell; gas-liquid interface medium, Promocell; or S-ALITM small airway gas-liquid interface medium, BulletKit™, LONZA, and any combination thereof.

[0034] In at least one embodiment of the present disclosure, the wafer may comprise: a second culture medium filling a second channel; an air layer formed in a first channel; and a gas-liquid interface formed between the second culture medium and the air layer.

[0035] In at least one embodiment of this disclosure, the membrane may be disposed between the gas-liquid interface and the second culture medium in the second channel.

[0036] In at least one embodiment of this disclosure, the first electrode, the second electrode and / or the third electrode may contact the gas-liquid interface and the region between the membrane.

[0037] In at least one embodiment of this disclosure, the first electrode may be a reference electrode, the second electrode may be a working electrode, and the third electrode may be a counter electrode; or the first electrode may be a working electrode, the second electrode may be a reference electrode, and the third electrode may be a counter electrode.

[0038] In at least one embodiment of this disclosure, the reference electrode, working electrode and / or relative electrode may comprise gold (Au), silver / silver chloride (Ag / AgCl), poly(3,4-ethylenedioxythiophene), poly(styrene sulfonate), platinum (Pt), carbon (C), graphene oxide, reduced graphene oxide and / or annealed graphene oxide.

[0039] In at least one embodiment of this disclosure, the microfluidic cell culture device may further include microparticles distributed in the first channel and / or the second channel.

[0040] In at least one embodiment of this disclosure, the cell may be selected from at least one of the group consisting of primary human small airway epithelial cells, primary human small airway epithelial cells-chronic obstructive pulmonary disease cells, NuLi-1 cells, CuFi-1 cells, Calu-3 epithelial cells, A549 cells and human intestinal epithelial cells.

[0041] In at least one embodiment of the present disclosure, the method of culturing cells may further include: providing a microfluidic cell culture device; introducing cells and a first culture medium to a membrane; and replacing the first culture medium with a second culture medium.

[0042] In at least one embodiment of the present disclosure, the method of culturing cells may further include: introducing a first culture medium to fill a first channel and / or a second channel.

[0043] In at least one embodiment of this disclosure, the method of culturing cells may further include: replacing the first culture medium with a second culture medium. The second culture medium may fill a second channel; an air layer may be formed in the first channel; and an air-liquid interface may be formed between the second culture medium and the air layer.

[0044] In at least one embodiment of this disclosure, the membrane may be disposed between the gas-liquid interface and the second culture medium in the second channel.

[0045] In at least one embodiment of this disclosure, the method of culturing cells may further include: measuring the electrochemical impedance spectrum of the cells.

[0046] In at least one embodiment of this disclosure, the method of culturing cells may further include: determining the cell physiology of the cells based on electrochemical impedance spectroscopy.

[0047] Figure 1A shows a schematic diagram of the first substrate 100 of a microfluidic cell culture apparatus 1 according to an embodiment of the present disclosure, including a first channel 1000, a first trench 1001 extending from the first channel 1000 and having a first opening 1011 formed at the edge of the first substrate 100, a second trench 1002 extending from the first channel 1000 and having a second opening 1012 formed at the edge of the first substrate 100, and a third trench 1003 extending from the first channel 1000 and having a third opening 1013 formed at the edge of the first substrate 100. It should be noted that the number and connection method of each component are illustrative and can be increased, decreased, or adjusted according to actual needs. In some embodiments of the present disclosure, the first substrate 100 may be, but is not limited to, a polycarbonate substrate and / or a polydimethylsiloxane plate. In some embodiments disclosed herein, the length of the first substrate 100 may be 20 mm to 128 mm; the width of the first substrate 100 may be 16 mm to 86 mm; and the height of the first substrate 100 may be 2 mm to 15 mm, but this disclosure is not limited thereto.

[0048] The upper left, lower left, and right views of Figure 1B show schematic diagrams of the second substrate 101 of the microfluidic cell culture apparatus 1 according to one embodiment of the present disclosure, including a second channel 1010. It should be noted that the number and connection method of each component are illustrative and can be increased, decreased, or adjusted according to actual needs. In some embodiments of the present disclosure, the second substrate 101 may be, but is not limited to, a polycarbonate substrate and / or a polydimethylsiloxane plate.

[0049] Figure 2 shows a top view of a first substrate 100 according to one embodiment of the present disclosure, including a first channel 1000, a first electrode 104, a second electrode 105, and a third electrode 106. It should be noted that the number and connection method of each component are illustrative and can be increased, decreased, or adjusted according to actual needs. In some embodiments of the present disclosure, the first substrate 100 may be, but is not limited to, a polycarbonate substrate and / or a polydimethylsiloxane plate. In some embodiments of the present disclosure, the first electrode 104 may be a reference electrode and can be used to contact cells; the second electrode 105 may be a working electrode and can be used to contact cells; and the third electrode 106 may be a relative electrode and can be used to contact cells. In some embodiments of the present disclosure, the first electrode 104 may be a working electrode and can be used to contact cells; the second electrode 105 may be a reference electrode and can be used to contact cells; and the third electrode 106 may be a relative electrode and can be used to contact cells.

[0050] Figure 3 shows a top view of the wafer 10 of a microfluidic cell culture apparatus 1 according to one embodiment of the present disclosure, including a first substrate 100, a second substrate 101, a first channel 1000, a second channel 1010, a first electrode 104, a second electrode 105, and a third electrode 106. It should be noted that the number and connection method of each component are illustrative and can be increased, decreased, or adjusted according to actual needs. In some embodiments of the present disclosure, the first substrate 100 and the second substrate 101 may be, but are not limited to, a polycarbonate substrate and / or a polydimethylsiloxane plate. In some embodiments of the present disclosure, the first electrode 104 may be a reference electrode and can be used to contact cells; the second electrode 105 may be a working electrode and can be used to contact cells; and the third electrode 106 may be a counter electrode and can be used to contact cells. In some embodiments disclosed herein, the first electrode 104 may be a working electrode and may be used to contact cells; the second electrode 105 may be a reference electrode and may be used to contact cells; and the third electrode 106 may be a relative electrode and may be used to contact cells.

[0051] Figure 4 shows an exploded view of the wafer 10 of a microfluidic cell culture apparatus 1 according to one embodiment of the present disclosure, including a first substrate 100, a first trench 1001, a second trench 1002, a third trench 1003, a first opening 1011, a second opening 1012, a third opening 1013, a second substrate 101, a first channel 1000, a second channel 1010, a first electrode 104, a second electrode 105, a third electrode 106, and a membrane 102. It should be noted that the number and connection method of each component are illustrative and can be increased, decreased, or adjusted according to actual needs. In some embodiments of the present disclosure, the first substrate 100 and the second substrate 101 may be, but are not limited to, a polycarbonate substrate and / or a polydimethylsiloxane plate. In some embodiments of the present disclosure, the first electrode 104 may be a reference electrode and can be used to contact cells; the second electrode 105 may be a working electrode and can be used to contact cells; and the third electrode 106 may be a counter electrode and can be used to contact cells. In some embodiments disclosed herein, the first electrode 104 may be a working electrode and may be used to contact cells; the second electrode 105 may be a reference electrode and may be used to contact cells; and the third electrode 106 may be a relative electrode and may be used to contact cells. In some embodiments disclosed herein, the membrane 102 may be, but is not limited to, a porous polyethylene terephthalate membrane.

[0052] In some embodiments of this disclosure, the wafer 10 of the microfluidic cell culture device 1 is designed using the computer-aided design software SolidWorks (Dassault Systemes SA, France) and is manufactured by injection molding using polycarbonate (PC). It contains two parallel channels, namely, a first channel 1000 and a second channel 1010 (width 1,000 μm × height 200 μm). In some embodiments of this disclosure, the width of the first channel 1000 of the wafer 10 may be 0.1 mm to 2 mm; and the height of the first channel 1000 of the wafer 10 may be 0.1 mm to 2 mm, but this disclosure is not limited thereto.

[0053] In some embodiments of this disclosure, the wafer 10 of the microfluidic cell culture device 1 disclosed herein is made of polydimethylsiloxane (PDMS) by replica molding.

[0054] In some embodiments of this disclosure, the first groove 1001, the second groove 1002, and the third groove 1003, which respectively accommodate the first electrode 104, the second electrode 105, and the third electrode 106, can be cut after the injection molding process. In some embodiments of this disclosure, the length of the first groove 1001, the second groove 1002, and the third groove 1003 can be from 7.5 mm to 42.5 mm; the width of the first groove 1001, the second groove 1002, and the third groove 1003 can be from 0.1 mm to 1 mm; and the height of the first groove 1001, the second groove 1002, and the third groove 1003 can be from 0.1 mm to 1 mm, but this disclosure is not limited thereto.

[0055] In some embodiments of this disclosure, membrane 102, for example, a porous polyethylene terephthalate (PET) membrane, separates the first channel 1000 and the second channel 1010, forming a small airway and a microvascular channel, respectively. In some embodiments of this disclosure, the diameter of the pores in membrane 102 may be from 0.4 μm to 8 μm, but this disclosure is not limited thereto.

[0056] In some embodiments disclosed herein, the first substrate 100 (i.e., PC), the second substrate 101 (i.e., PC), and the film 102 are assembled by solvent bonding; in short, the first substrate 100 (i.e., PC), the second substrate 101 (i.e., PC), and the film 102 are first activated in oxygen plasma (60 W, 500 mTorr) for 2 minutes; the first substrate 100 (i.e., PC) and the second substrate 101 (i.e., PC) are then immersed in 3% (v / v) 95%... The membrane 102 was immersed in an ethanol solution of (3-glycidyloxypropyl)trimethoxysilane (Glymo) (Sigma, USA) for 1 hour; and immersed in a 3% (v / v) isopropanol (IPA) solution of (3-aminopropyl)triethoxysilane (APTES) (Sigma, USA) for 30 minutes. The membrane 102 was rinsed with 100% IPA solution and dried in an oven at 80°C for 30 minutes, and then clamped between the first channel 1000 of the aligned first substrate 100 (i.e., PC) and the second channel 1010 of the second substrate 101 (i.e., PC); and a compression force of 0.9 N / m was applied to the assembled wafer 10 of the microfluidic cell culture device 1 disclosed herein at 80°C for 1 hour to complete the solvent bonding process.

[0057] In some embodiments disclosed herein, the first substrate 100 (i.e., PDMS), the second substrate 101 (i.e., PDMS), and the membrane 102 are assembled by solvent bonding; in short, the first substrate 100 (i.e., PDMS), the second substrate 101 (i.e., PDMS), and the membrane 102 are first activated in oxygen plasma (60 W, 500 mTorr) for 2 minutes; and the membrane 102 is immersed in a 3% (v / v) solution of (3-Aminopropyl)triethoxysilane (APTES) (Sigma, USA) in isopropanol (IPA) for 30 minutes. The membrane 102 was rinsed with 100% IPA solution and dried in an oven at 80°C for 30 minutes. It was then sandwiched between the first channel 1000 of the aligned first substrate 100 (i.e., PDMS) and the second channel 1010 of the second substrate 101 (i.e., PDMS). A compressive force of 0.9 N / m was applied to the assembled wafer 10 of the microfluidic cell culture device 1 of the present disclosure at 60°C overnight to complete the solvent bonding process.

[0058] In some embodiments disclosed herein, an extracellular matrix (ECM) coating solution, such as, but not limited to, an apical solution, is applied to the first channel 1000 to coat the ECM, thereby promoting cell adhesion to the surface of the membrane 102; the wafer 10 is cultured overnight in a culture dish at 4°C to allow it to begin to solidify; the ECM coating solution is removed, and the first channel 1000 and the second channel 1010 are gently washed with DPBS, and then cells 107 and the first culture medium 108 are introduced; the cells 107 are digested with trypsin, for example, primary human small airway epithelial cells (HSAECs) (ATCC, PCS-301-010) or A549 cells (ATCC, CCL-185), and then seeded in the first culture medium 108 at a concentration of 2.5 × 10⁶ cells / mL, followed by incubation at 37°C and 5%... Incubate under static CO2 conditions for 4 to 6 hours to promote cell attachment; change the top medium of the first medium 108 daily to remove unattached cells; and keep the cell culture submerged until the cells are fully confluent.

[0059] Figures 5A and 5B respectively show schematic side views of the wafer 10 of the microfluidic cell culture apparatus 1 under electrochemical impedance spectroscopy, before and after the first culture medium 108 is replaced with the second culture medium 109, according to one embodiment of the present disclosure. The wafer 10 may include a first substrate 100, a first channel 1000, a second channel 1010, a second substrate 101, a first electrode 104, a second electrode 105, a third electrode 106, a gas-liquid interface 111, cells 107, a membrane 102, a first culture medium 108, and / or a second culture medium 109. Referring to Figure 5A, cells 107 are introduced into the membrane 102, and the first culture medium 108 is introduced to fill the first channel 1000 and / or the second channel 1010. The first culture medium 108 in the first channel 1000 or the second channel 1010 may be removed and / or replaced with the second culture medium 109. The first electrode 104, the second electrode 105, and / or the third electrode 106 can contact the cell 107 to measure the electrochemical impedance spectroscopy of the cell 107, and the cell physiology of the cell 107 can be determined based on the measured electrochemical impedance spectroscopy of the cell 107. After replacing the culture medium, referring to Figure 5B, the second culture medium 109 fills the second channel 1010 and can contact the cell 107 through the porous membrane 102. In the first channel 1000, an air layer is formed above the cell 107 cultured on the membrane 102, and an air-liquid interface 111 is formed between the air layer and the second culture medium 109, and contacts the cell 107 on the membrane 102. In the second channel, the membrane 102 is disposed between the air-liquid interface 111 and the second culture medium 109. The first electrode 104, the second electrode 105, and / or the third electrode 106 can contact the area between the air-liquid interface and the membrane, and can contact the cell 107 to measure the electrochemical impedance spectroscopy of the cell 107. In some embodiments disclosed herein, an air-liquid interface (ALI) 111 is established by removing the first culture medium 108 from the first channel 1000 and / or the second channel 1010, and replacing the first culture medium 108 (e.g., EX Plus medium) with a second culture medium 109 (e.g., PneumaCult™-ALI medium, StemCell; air-liquid interface medium, Promocell, C-21080; or S-ALITM small airway air-liquid interface medium BulletKit™, LONZA) approximately 3 to 5 days after inoculation, to induce mucociliary differentiation of cells 107. The second culture medium 109 fills the second channel 1010. The apical surface of cells 107 (e.g., HSAECs) is washed weekly with DPBS to remove debris and mucus. During the 4-week cell culture period, a constant flow of the second culture medium 109 is continuously perfused into the second channel 1010 at 120 μL / h and 5% at 37°C. The cells were placed in a CO2 incubator (C11) and flow problems and cell morphology were monitored every 3 days.In some embodiments of this disclosure, electrochemical impedance spectroscopy (EIS) spectra of cells were measured under immersion conditions (Fig. 5A) in which both the first channel 1000 and the second channel 1010 were filled with the first culture medium 108. The measurement results are plotted in Fig. 9 (Chip_3) and Fig. 10 (immersion (Z) and immersion (phase)). In some embodiments of this disclosure, EIS spectra of cells were measured under ALI conditions (Fig. 5B) in which the first channel 1000 had an air layer and the second channel 1010 was filled with the second culture medium 109. The measurement results are plotted in Fig. 9 (Chip_1) and Fig. 10 (ALI (Z) and ALI (phase)). In some embodiments of this disclosure, the first substrate 100 and the second substrate 101 may be, but are not limited to, a polycarbonate substrate and / or a polydimethylsiloxane plate. In some embodiments of this disclosure, the first electrode 104 may be a reference electrode and may be used to contact cells; the second electrode 105 may be a working electrode and may be used to contact cells; and the third electrode 106 may be a counter electrode and may be used to contact cells. In some embodiments of this disclosure, the first electrode 104 may be a working electrode and may be used to contact cells; the second electrode 105 may be a reference electrode and may be used to contact cells; the third electrode 106 may be a relative electrode and may be used to contact cells. In some embodiments of this disclosure, the cell 107 may be, but is not limited to, primary human small airway epithelial cells, primary human small airway epithelial cells-chronic obstructive pulmonary disease cells, NuLi-1 cells, CuFi-1 cells, Calu-3 epithelial cells, A549 cells, and / or human intestinal epithelial cells, but this disclosure is not limited to these. In some embodiments of this disclosure, the membrane 102 may be, but is not limited to, a porous polyethylene terephthalate membrane. It should be noted that the number and connection method of each component are illustrative and may be increased, decreased, or adjusted according to actual needs.

[0060] Figure 6 is a graph of the equivalent circuit model of a cell according to one embodiment of the present disclosure and the fitting result of the measured electrochemical impedance spectroscopy. In some embodiments of the present disclosure, the first electrode 104, the second electrode 105, and the third electrode 106 are inserted from the side of the first channel 1000 to directly contact the cell layer, allowing current to pass through the cell barrier. In some embodiments of the present disclosure, a potentiostat 12 from PalmSens BV (PalmSens4) is used to record the electrochemical impedance spectroscopy; under gas-liquid interface conditions, a 10 mV sinusoidal excitation signal of 1 Hz to 1 MHz is periodically applied during cell culture to perform three-point impedance measurements; and the measured electrochemical impedance spectra are paired with the equivalent circuit model to allow for efficient acquisition of impedance and cell capacitance values.

[0061] Figure 7 shows a microfluidic cell culture apparatus 1, comprising: a wafer 10, an incubator 11, and a potentiostat 12. The wafer 10 is placed inside the incubator 11, while the potentiostat 12 is placed outside the incubator 11. The incubator 11 and the potentiostat 12 are connected to the wafer 10. The incubator 11 is used to provide a culture medium flow in the second channel of the wafer and to provide a stable cell culture environment with constant temperature and humidity; and the potentiostat is used to record the electrochemical impedance spectrum of the cells cultured in the wafer. In addition, the wafer 10 is connected to a peristaltic pump of the incubator 11. It should be noted that the number and connection method of each component are illustrative and can be increased, decreased, or adjusted according to actual needs.

[0062] The left, middle, and right images of Figure 8A illustrate different arrangements of the working electrode and the reference electrode in a cell-free microfluidic cell culture apparatus. Specifically, the tops of both the working electrode and the reference electrode are completely positioned in the first channel of the microfluidic cell culture apparatus (left image of Figure 8A; 1 (top exposed)); half of the top of the working electrode and the top of the reference electrode are positioned in the first channel of the microfluidic cell culture apparatus (middle image of Figure 8A; 0.5 (center)); or neither the top of the working electrode nor the top of the reference electrode is positioned in the first channel of the microfluidic cell culture apparatus (right image of Figure 8A; 0 (flat)). Furthermore, electrochemical impedance spectroscopy was performed on the working electrode and the reference electrode under different arrangements in the microfluidic cell culture apparatus (Figure 8B). Referring to Figure 8B, the measured electrochemical impedance is related to the distance between the tops of the working electrode and the tops of the reference electrode. The closer the tops of the working electrode and the tops of the reference electrode are, the lower the measured electrochemical impedance.

[0063] Figure 9 illustrates the difference in electrochemical cellular impedance measurement between the first and second channels of the wafer under different environmental arrangements; and provides four arrangements, including "Wafer_1" (a wafer containing a first channel with an air layer and a second channel with a liquid layer) (see Figure 5B); "Wafer_2" (a wafer containing a first channel with an air layer and a second channel with an air layer); "Wafer_3" (a wafer containing a first channel with a liquid layer and a second channel with a liquid layer) (see Figure 5A); and "Wafer_4" (a wafer containing a first channel with a liquid layer and a second channel with an air layer).

[0064] Referring to Figure 9, because the working electrode and reference electrode are disposed in the first channel of the wafer, the measured electrochemical cell impedance is significantly affected by environmental conditions. Since the resistance of the culture medium (i.e., the liquid layer) is much lower than that of the cells, the electrochemical cell impedance measured in wafers 1 and 2 (both with air layers in the first channel) is higher than that measured in wafers 3 and 4 (both with liquid layers in the first channel). Furthermore, according to Ohm's law, current is inversely proportional to resistance. Therefore, when the environment is filled with culture medium (i.e., the liquid layer) (wafers 3 and 4), the main path of the current is through the culture medium (i.e., the liquid layer) rather than the cells, resulting in lower measured electrochemical cell impedance results. On the other hand, the environmental arrangement of wafers 1 and 2 (both with air layers in the first channel) can reduce the measurement differences caused by the culture medium, thereby obtaining more accurate results. In wafer 2 (with air layers in both the first and second channels), the potential influence of the culture medium is completely eliminated, confirming that the signal measured by the microfluidic cell culture device does indeed pass through the cell layer and reach the electrode at the other end.

[0065] The upper image of Figure 10 shows a bright-field image of cells cultured in the first channel of a wafer in an ALI environment (see Figure 5B). The wafer arrangement for further measurement of electrochemical cell impedance includes: the top of the first electrode (e.g., working electrode or reference electrode) and the top of the second electrode (e.g., working electrode or reference electrode) are not disposed in the first channel; and half of the top of the third electrode (e.g., opposite electrode) is disposed in the first channel, but this disclosure is not limited thereto.

[0066] In some embodiments of this disclosure, the wafer arrangement for further measuring electrochemical cell impedance includes: half or all of the top of a first electrode (e.g., a working electrode or a reference electrode); half or all of the top of a second electrode (e.g., a working electrode or a reference electrode) disposed in a first channel; and half or all of the top of a third electrode (e.g., a counter electrode) disposed in the first channel.

[0067] In some embodiments of this disclosure, the wafer arrangement for further measuring electrochemical cell impedance includes: half or all of the top of a first electrode (e.g., a working electrode or a reference electrode); half or all of the top of a second electrode (e.g., a working electrode or a reference electrode) disposed in the first channel; and the top of a third electrode (e.g., a counter electrode) not disposed in the first channel.

[0068] Referring to the lower part of Figure 10, the electrochemical cell impedance and phase measurement results show that the microfluidic cell culture device can detect cell signals in an ALI environment (see Figure 5B). The results are significantly different from the results of the microfluidic cell culture device detecting cell signals in an immersion environment (see Figure 5A), confirming the applicability of the microfluidic cell culture device.

[0069] In some embodiments of this disclosure, the higher the cell density cultured in the microfluidic cell culture device, the higher the measured electrochemical impedance.

[0070] The above embodiments are merely illustrative and not intended to limit the scope of this disclosure. Those skilled in the art to which this disclosure pertains can modify and alter the above embodiments without departing from the spirit and scope of this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims, and all equivalent changes and modifications made in accordance with the claims of this disclosure shall fall within the scope of this disclosure. [Simplified Explanation of the Diagram]

[0013] These and other objects of this disclosure will be readily understood by those skilled in the art upon reading the following detailed description of embodiments illustrated in various different figures. The upper left view of FIG1A is a schematic top view of a first substrate of a microfluidic cell culture apparatus according to an embodiment of this disclosure. The lower left view of FIG1A is a schematic front view of the first substrate of a microfluidic cell culture apparatus according to an embodiment of this disclosure. The right view of FIG1A is a schematic side view of the first substrate of a microfluidic cell culture apparatus according to an embodiment of this disclosure. The upper left view of FIG1B is a schematic top view of a second substrate of a microfluidic cell culture apparatus according to an embodiment of this disclosure. The lower left view of FIG1B is a schematic front view of the second substrate of a microfluidic cell culture apparatus according to an embodiment of this disclosure. The right view of FIG1B is a schematic side view of the second substrate of a microfluidic cell culture apparatus according to an embodiment of this disclosure. FIG2 is an image of a top view of the first substrate of a microfluidic cell culture apparatus according to an embodiment of this disclosure. FIG3 is an image of a top view of a chip of a microfluidic cell culture apparatus according to an embodiment of this disclosure. Figure 4 is a schematic exploded view of a wafer of a microfluidic cell culture device according to an embodiment of the present disclosure. Figure 5A is a schematic side view of an electrochemical impedance spectroscopy (EIS) measurement of a wafer of a microfluidic cell culture device according to an embodiment of the present disclosure under an immersion environment. Figure 5B is a schematic side view of an EIS measurement of a wafer of a microfluidic cell culture device according to an embodiment of the present disclosure under an ALI environment. Figure 6 is a graph showing the fitting results of the equivalent circuit model of the cell and the measured electrochemical impedance spectroscopy according to an embodiment of the present disclosure. Figure 7 is a schematic diagram of a microfluidic cell culture device according to an embodiment of the present disclosure. The left image of Figure 8A is an image showing the arrangement of the first and second electrodes in the wafer under a cell-free state according to an embodiment of the present disclosure. The middle image of Figure 8A is an image showing the arrangement of the first and second electrodes in the wafer under a cell-free state according to an embodiment of the present disclosure. The right image of Figure 8A is an image showing the arrangement of the first and second electrodes in the wafer under a cell-free state according to an embodiment of the present disclosure. Figure 8B is a graph showing the electrochemical impedance spectroscopy measurements of the first and second electrodes in a wafer under different arrangements, according to an embodiment of the present disclosure ("Z" represents impedance; "freq" represents frequency). Figure 9 is a graph showing the electrochemical impedance spectroscopy measurements of the first and second channels in a wafer under different environmental arrangements, according to an embodiment of the present disclosure ("Z" represents impedance; "freq" represents frequency; "Wafer_1" represents a wafer containing a first channel with an air layer and a second channel with a liquid layer; "Wafer_2" represents a wafer containing a first channel with an air layer and a second channel with an air layer; "Wafer_3" represents a wafer containing a first channel with a liquid layer and a second channel with a liquid layer; "Wafer_4" represents a wafer containing a first channel with a liquid layer and a second channel with an air layer).The upper part of Figure 10 is a bright-field image of cells cultured in an ALI environment in an embodiment of the present disclosure. The lower part of Figure 10 is a graph of electrochemical impedance spectroscopy measurements in an ALI or immersion environment in an embodiment of the present disclosure ("Z" represents impedance; "freq" represents frequency; "ALI" represents gas-liquid interface; "neg. phase / 0" represents the phase difference between the input signal and the output signal).

Claims

1. A chip, comprising: A first substrate includes: a first channel; a first trench extending from the first channel and having a first opening formed on an edge of the first substrate; a second trench extending from the first channel and having a second opening formed on an edge of the first substrate; and a third trench extending from the first channel and having a third opening formed on an edge of the first substrate; and a second substrate coupled to the first substrate includes: a second channel corresponding to the first channel; and a membrane disposed between the first substrate and the second substrate.

2. The chip as described in claim 1, wherein, The first channel is coated with extracellular matrix.

3. The chip as described in claim 1, further comprising: The first electrode is partially disposed on the first trench; The second electrode is partially disposed on the second trench; And a third electrode, which is partially disposed on the third trench.

4. The chip as described in claim 3, wherein, At least a portion of the first electrode is disposed on the first trench through the first opening; at least a portion of the second electrode is disposed on the second trench through the second opening; and at least a portion of the third electrode is disposed on the third trench through the third opening.

5. The chip as described in claim 1, wherein, The membrane is porous and includes polyethylene terephthalate.

6. A microfluidic cell culture device, comprising: A wafer includes: a first substrate including: a first channel; a first trench extending from the first channel and having a first opening formed on an edge of the first substrate; a second trench extending from the first channel and having a second opening formed on an edge of the first substrate; a third trench extending from the first channel and having a third opening formed on an edge of the first substrate; a first electrode partially disposed on the first trench; a second electrode partially disposed on the second trench; and a third electrode partially disposed on the third trench; a second substrate coupled to the first substrate including: a second channel corresponding to the first channel; and a membrane disposed between the first substrate and the second substrate; an incubator housing the wafer to provide a culture medium flow in the second channel; and a potentiostat coupled to the first electrode, the second electrode, and the third electrode to record the electrochemical impedance spectroscopy of cells.

7. The apparatus as described in claim 6, wherein, At least a portion of the first electrode is disposed on the first trench through the first opening; at least a portion of the second electrode is disposed on the second trench through the second opening; and at least a portion of the third electrode is disposed on the third trench through the third opening.

8. The apparatus as described in claim 6, wherein, The wafer includes a first culture medium disposed on the first channel and / or the second channel.

9. The apparatus as described in claim 6, wherein, The wafer comprises: a second culture medium filling the second channel; an air layer formed in the first channel; and an air-liquid interface formed between the second culture medium and the air layer.

10. The apparatus as described in claim 9, wherein, The membrane system is disposed between the gas-liquid interface and the second culture medium in the second channel.

11. The apparatus as described in claim 10, wherein, The first electrode, the second electrode, and / or the third electrode contact the region between the gas-liquid interface and the membrane.

12. The apparatus as described in claim 11, wherein, The first electrode is a reference electrode, the second electrode is a working electrode, and the third electrode is a relative electrode; or wherein the first electrode is a working electrode, the second electrode is a reference electrode, and the third electrode is a relative electrode.

13. A method for culturing cells, comprising: Provide a microfluidic cell culture apparatus as described in claim 6; And the introduction of the cell into the membrane.

14. The method as described in claim 13 further includes introducing a first culture medium to fill the first channel and / or the second channel.

15. The method as described in claim 14, further comprising replacing the first culture medium with a second culture medium, wherein, The second culture medium fills the second channel; an air layer is formed in the first channel; And a gas-liquid interface is formed between the second culture medium and the air layer.

16. The method as described in claim 15, wherein, The membrane system is disposed between the gas-liquid interface and the second culture medium in the second channel.

17. The method as described in claim 16, wherein, The first electrode, the second electrode, and / or the third electrode contact the region between the gas-liquid interface and the membrane.

18. The method as described in claim 13, wherein, The cell line was selected from at least one of the following groups: primary human small airway epithelial cells, primary human small airway epithelial cells-chronic obstructive pulmonary disease cells, NuLi-1 cells, CuFi-1 cells, Calu-3 epithelial cells, A549 cells, and human intestinal epithelial cells.

19. The method as described in claim 13 further includes measuring the electrochemical impedance spectroscopy of the cell.

20. The method as described in claim 19 further includes determining the cell physiology of the cell based on the electrochemical impedance spectroscopy.

Images