Microfluidic apparatus and system, and method for culturing cells and / or tissues using same

Abstract

Provided is a microfluidic apparatus comprising: a first substrate having a first microfluidic channel, a second substrate having a second microfluidic channel, and a membrane. The membrane is arranged between the first substrate and the second substrate. Also provided is a microfluidic system comprising: the microfluidic apparatus, a first pump, and a second pump. The first pump can form a first closed loop with the microfluidic apparatus by means of a first pipeline. The second pump can form a second closed loop with the microfluidic apparatus by means of a second pipeline. Also provided is a method for culturing cells and / or tissues, comprising: providing the microfluidic system, and culturing the cells and / or tissues in the microfluidic apparatus.

Description

Microfluidic devices, systems and methods for culturing cells and / or tissues Technical Field

[0001] This disclosure relates to techniques for culturing cells and / or tissues, and more particularly to techniques for culturing cells and / or tissues using biomimetic in vitro models that combine diverse airflow fields and microchannel devices. Background Technology

[0002] In the field of clinical medical research, animal models are widely used in clinical drug development to explore the mechanisms of human health and disease, providing significant assistance in drug development and disease discovery. However, due to differences between animal models and humans in physiological structure, pathogenesis, and gene expression, drugs often fail when they enter clinical trials after completing animal testing. Furthermore, with rising awareness of animal welfare, the use of laboratory animals in research may raise ethical concerns. To address these issues, static in vitro models for cell culture have been developed, reducing the need for laboratory animals in drug development experiments and providing a more reliable research platform. Organ-on-a-chip (OoC) is a three-dimensional in vitro modeling technology integrating microfluidics. Its core concept involves culturing cells in a tiny platform, using microfluidics to achieve dynamic cell culture and provide the nutrients needed for cell growth, thereby establishing three-dimensional cellular tissues to reproduce organ function and more realistically simulate the internal environment of the human body. Organ-on-a-chips facilitate in-depth research into cellular behavior and physiological responses within the physiological microenvironment, and are of great significance not only for drug development but also for disease research and other biological research.

[0003] Currently, microfluidics technology is quite mature and widely used in research fields such as biomedicine, clinical diagnostics, and chemical analysis. However, most existing microchannels can only generate a single flow field characteristic, making it impossible to further explore the correlation between different flow field characteristics and analytical targets. On the other hand, organ-on-a-chip based on microfluidics technology can simulate the physiological microenvironment within a single human body (such as the respiratory tract, intestines, and stomach) to deeply explore the behavior of cells cultured on organ-on-a-chip, including cell proliferation, cell differentiation, and cell metabolism. However, the structures of the human respiratory tract, intestines, and stomach are mostly irregular, and the properties of the fluid flow field are altered by the organ and tissue structure when fluid passes through the organ. Current organ-on-a-chip microchannels are mostly of a single form, which cannot meet the diverse organ and tissue structures of the human body. In addition, culturing lung-related cells (such as human small airway ciliated cells) using organ-on-a-chip based on microfluidics technology typically requires 21 to 28 days. The long culture time makes data acquisition difficult, and organ-on-a-chip based on microfluidics technology can only provide a single culture condition at a time. Therefore, there is currently a lack of methods to accelerate the culture of lung-related cells and increase the throughput of organ-on-a-chip.

[0004] In summary, although there have been many drug development studies using organ-on-a-chip in recent years, culturing lung-related cells using organ-on-a-chip still requires a lot of time and money. Therefore, there is an urgent need in this field for a microfluidic device and system for culturing lung-related cells to accelerate the differentiation of lung-related cells and reduce the time and money costs of lung-related drug development.

[0005] Summary of the Invention

[0006] To address the aforementioned problems, this disclosure provides a microfluidic device comprising: a first substrate having a first microchannel, wherein the first microchannel has: a first flow channel having a first open end; a second flow channel having a second open end; and a third flow channel between the first flow channel and the second flow channel, wherein the third flow channel has a narrow section, and the average width of the narrow section is less than the average width of the third flow channel; a second substrate having a second microchannel, wherein the second microchannel has: a fourth flow channel having a third open end; a fifth flow channel having a fourth open end; and a sixth flow channel between the fourth flow channel and the fifth flow channel; and a thin film disposed between the first substrate and the second substrate and connected to the first substrate and the second substrate; wherein the third flow channel corresponds to the sixth flow channel, the first open end does not correspond to the third open end, and the second open end does not correspond to the fourth open end.

[0007] This disclosure also provides a microfluidic system comprising: the microfluidic device described in this disclosure; a first pump that forms a first closed loop with the microfluidic device via a first pipeline for supplying a first fluid to flow in the first closed loop; and a second pump that forms a second closed loop with the microfluidic device via a second pipeline for supplying a second fluid to flow in the second closed loop.

[0008] This disclosure also provides a method for culturing cells and / or tissues, comprising: the microfluidic system described in this disclosure; and culturing cells and / or tissues in a microfluidic device. Attached Figure Description

[0009] Figure 1 is a schematic diagram of the third flow channel of a microfluidic device according to one specific embodiment of the present disclosure.

[0010] Figure 2 is a flowchart of the fabrication of a first substrate and a second substrate of a microfluidic device according to one specific embodiment of this disclosure.

[0011] Figure 3 is a flowchart of establishing an in vitro model of a biomimetic human lung small airway according to one specific embodiment of this disclosure.

[0012] The upper part of Figure 4A is a schematic diagram of a long, straight microfluidic device according to one specific embodiment of the present disclosure; the lower part of Figure 4A is a schematic diagram of a microfluidic device with a narrow section according to one specific embodiment of the present disclosure.

[0013] The upper part of Figure 4B is an immunofluorescence staining image of the deposition distribution of gas-exposed fluorescent particles in a long, straight microfluidic device according to a specific embodiment of the present disclosure; the lower part of Figure 4B is an immunofluorescence staining image of the deposition distribution of gas-exposed fluorescent particles in a microfluidic device with a narrow section according to a specific embodiment of the present disclosure.

[0014] Figure 4C is a histogram showing the deposition distribution of gas-exposed fluorescent particles in a microfluidic device with a narrow section according to one specific embodiment of the present disclosure.

[0015] Figure 5A is a schematic diagram of a microfluidic system according to one specific embodiment of the present disclosure.

[0016] Figure 5B is a physical diagram of a microfluidic system according to one specific embodiment of this disclosure.

[0017] Figure 6 is a bright-field diagram of the cell state of cells cultured in a microfluidic device under different airflow field environments according to one specific embodiment of this disclosure.

[0018] Figure 7 is an immunofluorescence staining image of cell viability of cells cultured in a microfluidic device under different airflow field environments according to one specific embodiment of the present disclosure.

[0019] Figure 8A is a wind rose diagram of mucus and cilia removal in a long, straight microfluidic device cultured under different airflow field environments according to one specific embodiment of this disclosure.

[0020] Figure 8B is a wind rose diagram of mucus and cilia removal in a microfluidic device with a narrow opening, cultivated under different airflow field environments according to one specific embodiment of the present disclosure.

[0021] Figure 8C is a histogram of mucociliary clearance of a microfluidic device or a microfluidic device with a narrow section cultured in different airflow field environments according to a specific embodiment of the present disclosure.

[0022] Figure 9A is a wind rose diagram showing the directionality of cilia in a long, straight microfluidic device cultivated under different airflow field environments according to one specific embodiment of this disclosure.

[0023] Figure 9B is a wind rose diagram showing the directionality of cilia in a microfluidic device with a narrow section, cultivated under different airflow field environments according to one specific embodiment of this disclosure.

[0024] Figure 10A is an immunofluorescence staining image of small airway epithelial cell differentiation under different airflow field environments according to one specific embodiment of this disclosure.

[0025] Figure 10B is a histogram showing the differentiation of small airway epithelial cells under different airflow field environments according to one specific embodiment of this disclosure.

[0026] The upper left portion of Figure 11 is a thermal diagram of the cilia oscillation frequency of a long, straight microfluidic device under different airflow field environments according to a specific embodiment of the present disclosure; the lower left portion of Figure 11 is a thermal diagram of the cilia oscillation frequency of a microfluidic device with a narrow section under different airflow field environments according to a specific embodiment of the present disclosure; the right portion of Figure 11 is a histogram of the cilia oscillation frequency of a long, straight microfluidic device and / or a microfluidic device with a narrow section under different airflow field environments according to a specific embodiment of the present disclosure.

[0027] The upper part of Figure 12 is a top view of the mucus layer of a microfluidic device with a narrow opening according to a specific embodiment of the present disclosure; the lower part of Figure 12 is a side view of the mucus layer of a microfluidic device with a narrow opening according to a specific embodiment of the present disclosure.

[0028] The left part of Figure 13 is an immunofluorescence staining image of the mucus layer thickness of a long, straight microfluidic device and / or a microfluidic device with a narrow section under different airflow field environments according to a specific embodiment of the present disclosure; the right part of Figure 13 is a histogram of the mucus layer thickness of a long, straight microfluidic device and / or a microfluidic device with a narrow section under different airflow field environments according to a specific embodiment of the present disclosure. Detailed Implementation

[0029] The following specific embodiments illustrate the implementation of this disclosure. Those skilled in the art can easily understand the spirit, advantages, and effects of this disclosure based on the content contained herein. However, the specific embodiments described herein are not intended to limit this disclosure. This disclosure can also be implemented or applied through other different implementation methods, and the details described herein can also be given different changes or modifications according to different viewpoints and applications without departing from the spirit of this disclosure.

[0030] The proportions, structures, sizes, and other features shown in the accompanying drawings are only for illustrative purposes and to enable those skilled in the art to read and understand this disclosure. They are not intended to limit the scope of this disclosure. Therefore, any changes in proportions, modifications to structures, or adjustments to sizes, without affecting the purpose or effects of this disclosure, should fall within the scope of the technical content disclosed herein.

[0031] When the terms "include," "comprise," or "have" a specific element are used in this document, unless otherwise stated, other components, parts, structures, regions, sections, locations, devices, systems, steps, modules, or connections may be included, rather than excluding, such other elements.

[0032] The terms "upper," "lower," "front," and "rear" used in this document are only for illustrating specific embodiments of this disclosure and are not intended to limit the scope of implementation of this disclosure. Any adjustment, interchange, or change of their relative positions and relationships should be considered as part of the scope of implementation of this disclosure without substantially altering its technical content.

[0033] The terms "first" and "second" used herein are for the purpose of describing or distinguishing components, parts, structures, regions, sections, parts, devices, systems, modules, etc., and are not intended to limit the scope of this disclosure or to limit the spatial order of such components. Furthermore, unless otherwise expressly stated herein, the singular forms "a" and "the" used herein also include the plural forms, and the terms "or" and "and / or" used herein are interchangeable.

[0034] The numerical ranges described herein are inclusive and combinable. Any value falling within the numerical ranges described herein can be used as a maximum or minimum value to derive a subrange. For example, the numerical range of "4 hours to 6 hours" should be understood to include any subrange between the minimum value of 4 hours and 6 hours, such as: 4 hours to 4.5 hours, 4.2 hours to 5.5 hours, and 5 hours to 5.5 hours, etc. Furthermore, the multiple numerical endpoints described herein can be arbitrarily chosen as maximum or minimum values ​​to derive a numerical range; for example, 4 hours, 4.5 hours, and 6 hours can derive numerical ranges of 4 hours to 4.5 hours, 4.5 hours to 6 hours, or 4 hours to 6 hours.

[0035] In some embodiments, unless otherwise expressly stated herein, the terms “organ-on-a-chip,” “microfluidic device,” “microfluidic-based organ-on-a-chip,” “microfluidic,” “microfluidic device,” and / or “chip” are used interchangeably.

[0036] In some embodiments, unless otherwise expressly stated herein, the terms “small airway epithelial cells,” “small airway bronchial epithelial cells,” “small airway tissue,” and / or “small airway cells” may be used interchangeably.

[0037] In some embodiments, unless otherwise expressly stated herein, the terms “culture medium” and / or “culture medium” are used interchangeably.

[0038] Materials and Methods

[0039] Fabrication of long, straight microfluidic devices

[0040] The microfluidic device has a two-layer structure, comprising a first substrate (e.g., but not limited to, a polycarbonate (PC) plastic sheet) with a first microchannel, a second substrate (e.g., but not limited to, a polycarbonate plastic sheet) with a second microchannel, and a porous polyester membrane (PETE membrane) (Transparent, 0.4 micron, 12 micron thickness, Sterlitech / USA), with the first and second substrates separated by the PETE membrane. First, the PETE membrane, the first substrate, and the second substrate are modified with oxygen plasma. Then, the PETE membrane is immersed in isopropanol (IPA) containing 3% (3-Aminopropyl)triethoxysilane (APTES) for 30 minutes, followed by immersion in 100% isopropanol for 3 minutes to remove excess chemical bonds. Finally, the PETE membrane is dried in an 80°C oven for 40 minutes and then stored in alcohol. In addition, the first substrate and the second substrate were immersed in anhydrous alcohol containing 3% Glymo for 60 minutes. After the bonding reaction on the surfaces of the first substrate and the second substrate was completed, the first substrate, the PETE film and the second substrate were tightly assembled. The assembled microfluidic device with a double-layer structure was placed and clamped with iron plates and locked with screws. It was then placed in an 80°C oven to dry for 45 minutes to facilitate the evaporation of chemical agents in the microchannels.

[0041] Fabrication of microfluidic devices with narrow sections

[0042] In some embodiments, this disclosure provides a microfluidic device (see FIG1) with an inwardly recessed (1 mm wide, but not limited thereto) in the third flow channel MC3 (width may be 2 mm, but this disclosure is not limited thereto) of the first microfluidic channel, the recess being formed by a protrusion 1200A of the third flow channel MC3. As shown in Figure 2, after designing a narrow constriction in the third section MC3 of the first microfluidic channel in the mold of the microfluidic device, the A and B components of polydimethylsiloxane (PDMS) are uniformly mixed in a 10:1 ratio (Step 1; S1). Air bubbles in the PDMS mixture are removed using a vacuum pump (Step 2; S2). The mixture is then poured into an acrylic mold (Steps 3 to 4; S3 to S4), and cured in a 60°C oven for 24 hours (Step 5; S5). Finally, it is demolded (Step 6; S6) to obtain the first substrate 120 and the second substrate 122 of this disclosure. The modification method of the PETE film of the microfluidic device with the narrow constriction is the same as the modification method of the PETE film of the long, straight microfluidic device described above. However, the chemical modification method of the first substrate 120 or the second substrate 122 including PDMS is different from that of the first substrate or the second substrate of the long and straight microfluidic device. The first substrate 120 or the second substrate 122 including PDMS does not need to be modified by any chemical agents. The first substrate 120 and / or the second substrate 122 including PDMS are directly cleaned and dried. Finally, after the surface of the first substrate 120 and / or the second substrate 122 is modified by an oxygen plasma machine, the first substrate 120 including PDMS, the PETE film and the second substrate 122 are immediately bonded together and placed on a heating plate to dry, thus completing the preparation of the microfluidic device with narrow space.

[0043] Establishment of a multi-mode airflow field system

[0044] In some embodiments, as shown in FIG5A, an airflow system is established using a first pump 11A (e.g., an industrial peristaltic pump (G100-1J, Longer Precision Pump Co., Ltd., but not limited thereto) to generate a specific airflow rate by adjusting the rotational speed of the first pump 11A, thereby introducing the airflow into the microfluidic device 12. First, a sensor 13 is used to measure the airflow rate output by the first pump 11A at different rotational speeds. The measurement results are then substituted into the wall stress calculation formula to convert them into the corresponding shear stress. The sensor includes an Arduino nano board and a flow meter (SENSOR AIRFLOW 200SCCM SHORT 5V, #HFBSF0200CX5, Honeywell Sensing and Productivity Solutions) connected to it. Given that the machine temperature of the first pump 11A is relatively high during continuous operation, if it is placed in the incubator 16, the internal temperature of the incubator 16 will be too high, causing the cells cultured in the microfluidic device 12 to die. Therefore, the first pump 11A is placed outside the incubator 16, and the first line 14A (e.g., a silicone peristaltic pump tube, but this disclosure is not limited thereto) is installed on the pump head of the first pump 11A, and the first line 14A is coupled to the first opening end O1 of the microfluidic device 12 inside the incubator.

[0045] Analysis of particle deposition distribution and quantification

[0046] In some embodiments, an air aeration system (SCIREQ) is used to aerate particles (with a particle size of 2 μm, but this disclosure is not limited thereto) containing red fluorescence (FluoSpheres). TM Carboxylate-modified microspheres (2.0 μm, red fluorescent (580 / 605), 2% solids) were exposed into the first microchannel of the microfluidic device. The aeration system parameters were set to a 5% duty cycle, a 1000 ms cycle time, and a 5-minute exposure time per exposure. After the particles were aerated into the first microchannel, an image of the entire first microchannel was captured using a fluorescence microscope. Then, the number of particles was calculated using software, and the result was divided by the area of ​​the first microchannel to calculate the particle density per unit area.

[0047] Establishment of an in vitro model of a biomimetic human lung small airway

[0048] In some embodiments, primary human small airway epithelial cells (HSAECs) derived from healthy humans (PCS–301–010, Information: Male, 16 years, Hispanic / Latino, LOT: 64079184, ATCC) are used to establish an in vitro model of the biomimetic human lung small airway, wherein the cell passage number used is between the 5th and 7th generation. After thawing, the HSAECs are first cultured in Ex-Plus medium (Pneumult). TM Cells (Ex plus Medium, STEMCELL #05040) were cultured in T75 tissue culture flasks until the cell density reached approximately 80%, at which point differentiation culture could begin. 24 hours before cell differentiation culture, deionized water and Collagen I (Thermo-Gibco, A1048301) were mixed at a 9:1 ratio to prepare a Collagen I dilution. This dilution was added to the first and second microchannels of the microfluidic apparatus and incubated overnight at 4°C. Before cell differentiation culture, the Collagen I dilution was aspirated from the first and second microchannels, and then the first and second microchannels were washed with Dulbecco's Phosphate-Buffered Saline (DPBS). Next, gently rinse the HSAECs twice with 4 mL of DPBS in a T75 tissue culture flask, then remove the DPBS. Add 3 mL of cell separation reagent (Trypsin 0.25% protease with porcine trypsin, HBSS, EDTA; without calcium, magnesium, 100 mL) and incubate for 5 minutes to allow the cells to react with the reagent and suspend in the T75 tissue culture flask. Add an equal volume of trypsin neutralizer (Trypsin Neutralizer / 100 mL, Gibco) to neutralize the cells. TM #R002100) Stop the separation reagent reaction in a T75 tissue culture flask. Collect the cell culture medium into a 15ml centrifuge tube and centrifuge for 5 minutes using a temperature-controlled centrifuge (Heraeus Megafuge 8, ThermoFisher) to separate the cells from the trypsin neutralization solution. After removing the trypsin neutralization solution, calculate the cell concentration and adjust the cell culture concentration to 2.5 x 10⁻⁶. 6Cell / ml. As shown in Figure 3, the cell solution, after concentration adjustment, is added (or seeded) into the first microchannel of the microfluidic device (Sub D0) and placed in an incubator for 4 to 6 hours to allow the cells to stably adhere to the first microchannel. After the cells in the first microchannel are stably attached, the culture medium in both the first and second microchannels is replaced, and unattached cells and metabolic waste are removed. Simultaneously, the culture medium in the second microchannel is switched to dynamic flow mode. Before switching the first microchannel to dynamic flow mode, all consumables must be sterilized. Then, the first microchannel is connected to the first pump and placed in an incubator using a micro-precision peristaltic pump for dynamic culture. The culture medium in the first microchannel is changed once daily to ensure sufficient nutrients for cell growth. On day 3 of immersion culture of HSAECs, after confirming the cell growth density and attachment status in the first microchannel, the cell culture mode is switched to air-liquid interface (ALI) differentiation culture (Sub D3). First, the culture medium in the first microchannel was drained, and then DPBS and ALI culture medium were used successively. TM Gently rinse the cell surface with ALI Medium (STEMCELL#05001), and replace the culture medium in the second microchannel with ALI medium. Continue to culture the cells in dynamic culture mode for 3 to 5 weeks (ALID21 to ALID28). During this period, rinse the cell surface with DPBS once a week to avoid the accumulation of mucus and cell debris, which may affect the cell differentiation ability.

[0049] Establishment of integrated airflow field system and microfluidic device

[0050] As shown in Figure 3, after 7 days of ALI differentiation culture and assessment of stable cell differentiation, HSAECs are fed with airflow into the first microchannel via the first pump (ALID7). To ensure sterility during airflow introduction, all consumables, including silicone peristaltic pump tubing, silicone tubing, adapters, Tryton tubing, and two 10mL glass sample vials (i.e., the first and second containment chambers of this disclosure), are autoclaved before airflow introduction. It should be noted that the two glass sample vials contain 5mL of DPBS to ensure a stable and sufficiently humidified airflow into the first microchannel. Until the ALI culture process is complete, the airflow system operates continuously except when replenishing new culture medium, which requires a temporary interruption of the airflow system (ALID28).

[0051] Verification of cell viability of cells cultured using the microfluidic system of this disclosure

[0052] Differentiated HSAECs were cultured continuously for two weeks after gas flow was introduced, and then analyzed using a cell viability kit (Cell-Check). TM Cell viability was determined using the Calein AM (AM) and Propidium (PI) methods from the Viability / Cytotoxicity Kit for Animal Cells. First, following the recommended staining concentrations on the product datasheet, PI and AM were diluted with DPBS to concentrations of 4 μM and 2 μM, respectively. Before adding the stains, the cell surface was rinsed with DPBS. Then, the stains were added to the first microfluidic channel, and the microfluidic apparatus containing the cells was returned to the incubator and incubated for 30 minutes. After staining, the cells were washed three times with DPBS, allowing the DPBS to remain in the first microfluidic channel for 5 minutes each time to ensure the removal of excess stain from the cell surface. Finally, the cells were fixed for 15 minutes using a Fixation / Permeabilization Solution (BD Cytofix / Cytoperm), washed twice more with DPBS, and images of the cells were captured using a fluorescence microscope or a high-throughput imaging system.

[0053] Real-time particle tracking imaging and analysis

[0054] In some embodiments, to evaluate the rate of mucociliary clearance (MCC) of mucous cells and ciliated cells differentiated from cells cultured using the microfluidic system of this disclosure for ALI, an air aeration system is used to aerate fluorescent particles (FluoSpheres). TMCarboxylate-modified microspheres (2.0 μm, red fluorescent (580 / 605), 2% solids) were exposed to differentiated human lung small airway tissue. The migration velocity of the fluorescent particles was observed under a microscope and recorded as images. Software was then used to analyze the quantified mucociliary clearance rate. First, fluorescent particles were mixed with DPBS at a 1:50 ratio and added to the reservoir chamber of the nebulizer in the aeration system. Simultaneously, the parameters of the aeration system were set, including airflow rate, airflow waveform, nebulizer duty cycle, and nebulizer cycle time. After parameter settings, the microfluidic device and nebulizer were connected, and the aeration system was started for a 5-minute particle exposure process. Immediately after exposure, images of particle movement within the microfluidic device were recorded using a fluorescence microscope (20x magnification) (image capture parameters: 536×536 pixels; exposure time: 10 ms). Then, software was used to analyze the particle trajectory and distance, calculating the direction and velocity of mucociliary clearance. In addition, the direction of particle movement is stored and the file is exported. Finally, the wind rose diagram is drawn using software to represent the direction of particle movement.

[0055] Analysis of Cilia Beating Frequency

[0056] On day 21 of ALI culture, differentiated human lung small airway tissue was cultured in ALI mode, and images of ciliary twitching were recorded using a 20x objective lens and a high-speed camera. Before recording, the image parameters were adjusted to an exposure time of 10ms and a frame rate of 100fps, with each 10-second video recording focusing on 8 to 10 different regions of the first microfluidic channel. Then, software was used to calculate the grayscale value change of each pixel in the images. The average pixel value was used as a threshold to filter out non-ciliary locations, and the grayscale value of each image in the remaining region was read to calculate the average ciliary twitching frequency in the field of view. The ciliary twitching frequency was presented as a heatmap, quantifying the ciliary twitching frequency in each field of view.

[0057] Immunofluorescence staining of differentiated small airway tissue cultured using the microfluidic system of this disclosure

[0058] In some embodiments, before staining differentiated small airway tissue, the cell tissue is gently rinsed with DPBS and fixed at room temperature for 15 minutes using a fixation / osmosis reagent, followed by rinsing the cell tissue twice with DPBS. To prevent antibodies (e.g., but not limited to anti-Ac-tubulin antibody, anti-MUC5B antibody, anti-Cytokeratin 5 antibody, or anti-Uteroglobin antibody) from binding to nonspecific proteins during staining, blocking buffer (1% BSA / 5% FBS) is added to the fixed cell tissue and allowed to stand for 30 minutes. Then, the primary antibody is diluted with DPBS at the recommended ratio and added to the cell tissue, and the tissue is incubated overnight at 4°C. The next day, the waste liquid in the microfluidic device containing the cell tissue treated with the primary antibody was removed. The microfluidic device was washed three times with DPBS. Then, the secondary antibody was diluted at the recommended ratio and added to the microfluidic device. The device was left to stand at room temperature for 2 hours to allow the primary and secondary antibodies to react fully. After the bonding reaction was completed, the waste liquid in the microfluidic device was aspirated. The sample was washed three times with DPBS. The PETE film with the cell tissue attached was picked up with tweezers and attached to a coverslip. The cell nuclei were counterstained with an anti-quenching agent (Antifade Mounting Medium with DAPI, H-1200, VECTASHIELD). Finally, the slide and coverslip were attached, and the cell tissue was imaged using a high-throughput imaging system and a conjugate fluorescence microscope.

[0059] Visualization and quantitative analysis of mucus thickness in differentiated human pulmonary small airway tissue

[0060] In some embodiments, to assess the mucus thickness of differentiated human pulmonary small airway tissue, cell tissue was fixed using Carnoy's fixative to preserve the mucus layer of the small airway tissue intact. Carnoy's fixative is a mixture of ethanol, chloroform, and acetic acid in a volume ratio of 6:3:1. To reduce mucus displacement and loss caused by shear stress during Carnoy's fixative injection into the first microchannel, the microchannel device was disassembled, the PETE membrane was removed, cells were fixed with Carnoy's fixative, and after standing for 5 minutes, waste liquid on the PETE membrane was removed. The remaining waste liquid on the PETE membrane was then washed away with DPBS, and this process was repeated twice. Next, primary antibody MUC5B (Anti-MUC5B antibody produced in rabbit, MERCK HPA008246) was added to the fixed cell tissue, and the tissue was placed at 4°C overnight to allow the target protein on the cell surface to fully react with the primary antibody MUC5B. The following day, the primary antibody waste liquid on the PETE membrane was aspirated, and the remaining waste liquid on the PETE membrane was washed away with DPBS, and this process was repeated three times. Next, secondary antibodies were added to the PETE film and allowed to stand for 2 hours. After the bonding time was complete, residual waste liquid on the PETE film was washed away with DPBS, and this process was repeated twice. Finally, the PETE film with attached cell tissue was picked up with forceps and attached to a coverslip containing DAPI mounting medium (Antifade Mounting Medium with DAPI, H-1200, VECTASHIELD). The slide and coverslip were then attached together, and the sides of the coverslip were sealed with nail polish. After visualizing the mucus in the differentiated human lung small airway tissue, the thickness of the mucus was photographed using a conjugate fluorescence microscope to quantify the amount of mucus secreted. Approximately 10 fields of view were selected using a 40x conjugate microscope to capture and output images. The mucus images were imported into software to analyze the average thickness of the mucus layer, and the results were statistically analyzed.

[0061] Analysis of the ciliary directionality of differentiated human pulmonary small airway tissue

[0062] To analyze the directionality of cilia in differentiated human pulmonary small airway tissue, firstly, the cilia matrix and footpiece in the cellular tissue were immunofluorescently stained with corresponding primary and secondary antibodies, respectively. Then, images of the cilia were taken using a 100x conjugate microscope at a resolution of 1024×1024. Since the footpiece and matrix of the cilia were still difficult to observe even with a 100x objective lens, the images taken with the 100x conjugate microscope were magnified 4.5 times before imaging. Next, the fluorescently stained images of the footpiece and matrix with red and green fluorescence were saved and exported. Software was used to analyze the directionality of ciliary movement, reading the images of the footpiece and matrix separately, calculating the area and centroid position of all target objects in the images, and simultaneously, using the centroid of each target object with red fluorescence as the origin, extending outwards with a radius of approximately 10 pixels, calculating the centroid of the closest target object with green fluorescence to the centroid of the target object with red fluorescence. Finally, the centroid coordinates of the target objects with green fluorescence and red fluorescence are used to calculate the vectors, and the images of the two are merged. The direction of the output vectors is plotted as a wind rose diagram. The direction of the arrows in the wind rose diagram represents the direction of the cilia's swing. It should be noted that the direction of the cilia's swing is determined by the base.

[0063] result

[0064] Example 1

[0065] Establish a biomimetic in vitro model of human lung small airways integrating airflow field system and microfluidic device

[0066] In some embodiments, the feasibility of the microfluidic system of this disclosure for cell culture is verified by comparing two microfluidic devices with different structures integrated with the airflow field system of this disclosure. The two microfluidic devices with different structures are (1) a conventional long straight microfluidic device for comparison (microfluidic channel width is 1 mm, but this disclosure is not limited thereto) (see the upper part of FIG4A; the upper part of FIG4A shows the third channel of the first microfluidic channel of the long straight microfluidic device) and (2) the microfluidic device of this disclosure with a narrow section (microfluidic channel width is 2 mm, and the third channel MC3 has an inwardly recessed protrusion of 1 mm width) (see the lower part of FIG4A; the lower part of FIG4A shows the third channel MC3 of the first microfluidic channel of the microfluidic device with a narrow section). In addition, this disclosure provides a microfluidic system 1 (i.e., a multi-modal airflow field system) to manufacture microfluidics that conform to / simulate the human airway environment (see FIG5A and FIG5B) to verify the feasibility of biomimetic microfluidics for cell culture. Furthermore, this disclosure provides a first accommodating chamber 15A and a second accommodating chamber 15B containing DBPS in a multi-mode airflow field system to provide stable airflow and maintain the humidity of the airflow. The first accommodating chamber 15A and the second accommodating chamber 15B containing DBPS are connected to the first opening end O1 of the first substrate 120 through a first pipeline 14A.

[0067] Functional verification of the structure of microfluidic devices

[0068] Figures 4A to 4C show the functional verification results of the microfluidic device structure. Fluorescent particles were aerated into the third channel MC3 of the first microfluidic channel using the aforementioned air aeration system, with the airflow direction from A to C (see the lower part of Figure 4A). The deposition location of the fluorescent particles was observed. The distribution of fluorescent particles in the third channel MC3 of the first microfluidic channel of the microfluidic device with a narrow section (see the lower part of Figure 4B) is less uniform compared to the third channel of the first microfluidic channel in a long, straight microfluidic device (see the upper part of Figure 4B). The lower part of Figure 4B shows the distribution density of fluorescent particles in different regions of the third channel MC3 of the first microfluidic channel. The highest distribution density of fluorescent particles is found at the narrow section formed by the protrusion 1200A (see Figure 4C). This indicates that the fluorescent particles are affected by the airflow field and the inertial collision mechanism within the first microfluidic channel during their movement, resulting in irregular movement and thus uneven distribution within the first microfluidic channel. Therefore, the model of the integrated airflow field system and the microfluidic device with narrow sections disclosed herein can not only generate diverse airflow fields, but also serve as a basis for exploring the functionality of small airway tissue in the human lungs.

[0069] Verification of long-term airflow supply using airflow field system

[0070] Figures 5A and 5B show the connection between the airflow field system and the microfluidic device 12. The airflow field system is established using a first pump 11A (e.g., an industrial peristaltic pump, but not limited thereto). The shaft of the first pump 11A is oriented and rotates at a constant speed, continuously delivering airflow into the microfluidic device 12. However, during the rotation of the shaft of the first pump 11A, the first pipeline 14A delivering the airflow is compressed, resulting in unstable airflow delivery. To solve the above problem, two first accommodating chambers 15A and 15B (e.g., sample vials, but not limited thereto) containing DPBS are added between the first pipeline 14A and the microfluidic device 12. This makes the airflow within the microfluidic device 12 more stable and simultaneously maintains the humidity of the airflow to prevent the cell culture environment within the microfluidic device 12 from becoming too dry.

[0071] Cell viability of cells cultured using the microfluidic system disclosed herein

[0072] Figure 6 shows the bright-field results of cells cultured for 2 weeks in the third channel of the first microfluidic channel under different airflow conditions (control group (no airflow; 0 ml / min), 3.5 ml / min, and 6 ml / min). Except for the right half of the third channel in the first microfluidic channel, where slight cell detachment occurs due to the significant shear stress generated the instant the airflow enters the first microfluidic channel, the cell adhesion in the rest of the third channel remains stable and without abnormalities. Figure 7 shows the cell viability results under different airflow conditions. Small airway cells can survive under airflow conditions of 3.5 mL / min and the control group; however, small airway cells show significant cell death under an airflow condition of 6 mL / min, indicating that cells cannot survive normally under the shear stress of an airflow condition of 6 mL / min during in vitro culture. Therefore, the culture method provided by this invention not only provides additional airflow for culture but also allows for continuous culture without significantly affecting cell conditions.

[0073] Example 2

[0074] Functional evaluation of human pulmonary small airway tissue cultured in the microfluidic device of this disclosure under different airflow environments.

[0075] Functional analysis of mucociliary clearance

[0076] Figures 8A to 8C show the functional evaluation results of mucociliary clearance (MCC) under different airflow environments. As shown in Figure 8A, in a static culture environment (no airflow), the MCCs of small airway tissues cultured in a long, straight microfluidic device move towards the lower right of the first microfluidic channel (i.e., directionally to the lower right). Conversely, in a culture environment with airflow (T), the MCCs of small airways move to the left of the first microfluidic channel in accordance with the direction of airflow, indicating that the mechanical stimulation generated by airflow has the ability to polarize the direction of the MCCs. As shown in Figure 8B, in an airflow environment, the MCCs of small airway tissues cultured in a microfluidic device with a narrow section in each region of the third channel MC3 of the first microfluidic channel (i.e., A (1 / 2T), B (narrow section), and C, where the three blue arrows in Figure 8B are schematic symbols for airflow) all have the same direction, and the clearance direction is almost consistent with the direction in the airless environment in Figure 8A. The clearance direction is also the same in B and C, where airflow disturbance is more likely to occur. As shown in Figure 8C, under the same culture environment, the MCC velocity of small airway tissue cultured in a long and straight microfluidic device is significantly higher than that of small airway tissue cultured in a microfluidic device with narrow sections, indicating that the consistency of MCC orientation may affect the MCC clearance rate.

[0077] Cilia directionality analysis

[0078] Figures 9A and 9B show the results of ciliary polarity direction under different airflow conditions (the three blue arrows in Figure 8B are schematic symbols for airflow). As shown in Figure 9A, in the absence of airflow, the direction of ciliary oscillation in the long, straight microfluidic device does not tend to be polarized. As shown in Figure 9B, after airflow is introduced into the microfluidic device with a narrow section, it is shown that the direction of the MCC is not significantly related to the directionality of ciliary oscillation. Therefore, it can be concluded that introducing gas into the first microchannel can increase the ciliary clearance speed without affecting the ciliary directionality.

[0079] Example 3

[0080] Cell differentiation of human pulmonary small airway bronchial epithelial cells cultured using the microfluidic system disclosed herein

[0081] In some embodiments, the effect of airflow intervention via a microfluidic device with narrow sections on cell differentiation of small airway bronchial epithelial cells is further verified.

[0082] Analysis of the cell differentiation types of small airway bronchial epithelial cells

[0083] Figures 10A and 10B show the cell differentiation results under different airflow conditions. As shown in Figure 10A, the differentiated cell types differed under different airflow conditions. Ac-tubulin is a biomarker for ciliated cells; MUC5B is a biomarker for goblet cells; ctyokeratin 5 is a biomarker for basal cells; and uteroglobin is a biomarker for secretory cells. As shown in Figure 10B, under airflow conditions, the number of cilia differentiated from small airway bronchial epithelial cells was less than that differentiated from small airway bronchial epithelial cells not cultured under airflow conditions. However, there was no significant difference in the number of goblet cells, basal cells, and secretory cells differentiated from small airway bronchial epithelial cells under different airflow conditions.

[0084] Analysis of Cilia Beating Frequency under Different Airflow Environments

[0085] Figure 11 shows the results of ciliary oscillation frequencies under different airflow conditions. As shown in the left part of Figure 11, after software analysis, the ciliary oscillation frequencies in the field of view are displayed as heatmaps. The upper part of the left part of Figure 11 shows the heatmaps of ciliary oscillation frequencies in microfluidic devices cultured under different airflow conditions; and the lower part of the left part of Figure 11 shows the heatmaps of ciliary oscillation frequencies in microfluidic devices with narrow sections cultured under different airflow conditions. As shown in the right part of Figure 11, the ciliary oscillation frequencies are approximately 9 to 10 Hz under different airflow conditions.

[0086] Analysis of mucus secretion from differentiated human pulmonary small airway tissue cultured in the microfluidic device of this disclosure, with or without airflow.

[0087] Figure 12 shows the immunofluorescence staining results of the mucus layer of differentiated human lung small airway tissue cultured in the microfluidic device of this disclosure. As shown in the upper part of Figure 12, Carnoy's fixative can completely preserve the structure of the mucus, which appears as a slender filamentous structure and an intact mucus layer in the field of view. As shown in the lower part of Figure 12, the thickness results after staining can be observed through the side view. Figure 13 shows the mucus layer thickness results of differentiated human lung small airway tissue under different airflow conditions. The left part of Figure 13 is the fluorescence staining image of the mucus layer thickness; and the right part of Figure 13 is the histogram of the mucus layer thickness. Even if the cell tissue is exposed to airflow for a long time, the amount of mucus secretion does not increase significantly. It can be seen that after the microfluidic device with narrow section of this disclosure is introduced with airflow, it can increase the amount of ciliary differentiation without affecting the function of other cells.

[0088] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

[0089] The reference numerals in the attached figures are explained as follows: 1: Microfluidic system; 10: Filter; 11A: First pump; 11B: Second pump; 12: Microfluidic device; 120: First substrate; 1200: First microchannel; 1200A: Protrusion; 121: Thin film; 122: Second substrate; 1220: Second microchannel; 13: Sensor; 14A: First pipeline; 14B: Second pipeline; 15A: First chamber; 15B: Second chamber; 16: Incubator; MC1: First flow channel; MC2: Second flow channel; MC3: Third flow channel; MC4: Fourth flow channel; MC5: Fifth flow channel; MC6: Sixth flow channel; O1: First opening end; O2: Second opening end; O3: Third opening end; O4: Fourth opening end.

[0090] [Biomaterial Storage]

[0091] none

Claims

1. A microfluidic device, characterized in that, comprising: a first substrate having a first microfluidic channel, wherein the first microfluidic channel has: a first segment of the channel having a first open end; a second segment of the channel having a second open end; and a third segment of the channel between the first segment of the channel and the second segment of the channel, wherein the third segment of the channel has a narrow segment and an average width of the narrow segment is less than an average width of the third segment of the channel; a second substrate having a second microfluidic channel, wherein the second microfluidic channel has: a fourth segment of the channel having a third open end; a fifth segment of the channel having a fourth open end; and a sixth segment of the channel between the fourth segment of the channel and the fifth segment of the channel; and a membrane disposed between and coupled to the first substrate and the second substrate; wherein the third segment of the channel corresponds to the sixth segment of the channel, the first open end does not correspond to the third open end, and the second open end does not correspond to the fourth open end.

2. The microfluidic device of claim 1, wherein, The third segment of the channel includes at least one protrusion to form the narrow segment.

3. The microfluidic device of claim 1, wherein, The first segment of the channel forms a first angle with the third segment of the channel, the second segment of the channel forms a second angle with the third segment of the channel, the fourth segment of the channel forms a third angle with the sixth segment of the channel, and the fifth segment of the channel forms a fourth angle with the sixth segment of the channel.

4. The microfluidic device of claim 1, wherein, The first segment of the channel does not correspond to the fourth segment of the channel, and the second segment of the channel does not correspond to the fifth segment of the channel.

5. A microfluidic system, characterized in that, comprising: the microfluidic device of claim 1; a first pump forming a first closed loop with the microfluidic device through a first line for a first fluid to flow in the first closed loop; and a second pump forming a second closed loop with the microfluidic device through a second line for a second fluid to flow in the second closed loop.

6. The microfluidic system of claim 5, wherein, Further comprising the first fluid and the second fluid, wherein the first fluid is a gas and the second fluid is a first liquid.

7. The microfluidic system of claim 6, wherein, The first liquid is located within the first microfluidic channel and / or the second microfluidic channel.

8. The microfluidic system of claim 6, wherein, The membrane is located at a gas-liquid interface of the gas and the first liquid.

9. The microfluidic system of claim 6, wherein, Further comprising cells and / or tissues located within the second microfluidic channel and / or at the gas-liquid interface of the gas and the first liquid.

10. The microfluidic system of claim 6, wherein, The first liquid is a culture medium.

11. The microfluidic system of claim 6, wherein, Further comprising at least one holding chamber disposed between the first pump and the microfluidic device to couple the first pump and the microfluidic device, wherein the holding chamber has a second liquid therein to maintain humidity of the gas flowing therethrough.

12. The microfluidic system of claim 11, wherein, Further comprising an incubator, wherein the microfluidic device and / or the holding chamber is disposed in the incubator.

13. The microfluidic system of claim 6, wherein, comprising: a first holding chamber coupled to the first pump and having a liquid therein to maintain humidity of the gas flowing therethrough; and a second holding chamber coupled to the first holding chamber and the first open end to stabilize flow of the gas. Further comprising a sensor coupled to the second open end.

14. The microfluidic system of claim 5, wherein, Further comprising a filter coupled to the first pump.

15. The microfluidic system of claim 5, wherein, ​ 16. The microfluidic system of claim 5, wherein, Also included are a filter, a first containment chamber, a second containment chamber, and a sensor, wherein the filter, the first pump, the first containment chamber, the second containment chamber, the microfluidic device, and the sensor are coupled by the first line to form the first closed loop.

17. The microfluidic system of claim 5, wherein, The second pump, the third opening, and the fourth opening are coupled by the second line to form the second closed loop.

18. A method of culturing cells and / or tissues, comprising, Providing a microfluidic system as claimed in claim 5; and Culturing cells and / or tissues in the microfluidic device. The microfluidic system also includes a first fluid and a second fluid, and the method also includes:

19. The method of claim 18, wherein, driving the first fluid to flow in the first closed loop by the first pump; and driving the second fluid to flow in the second closed loop by the second pump. The first fluid is a gas, the second fluid is a first liquid, the cells and / or the tissues are located within the second microfluidic channel and / or at a gas-liquid interface of the gas and the first liquid, and the gas flows through the narrow section.

20. The method of claim 19, wherein, ​

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