Microfluidic device for cell culture and method for manufacturing same

Abstract

Microfluidic devices and methods of making the same are provided. A microfluidic device (10) includes a base layer (100), a first layer (200) disposed on the base layer (100), and a second layer (300) disposed on the first layer (200). The first layer (200) defines one or more cell culture chambers (210). Each of the one or more cell culture chambers (210) is configured to contain one or more cells. The second layer defines one or more plenums (310). Each of the one or more gas chambers (310) is adjacent to a respective cell culture chamber (210). Microfluidic devices allow for the culture of single or more human-derived or animal-derived cells, or their co-culture with microorganisms, under static or dynamic culture conditions.

Description

Technical Field

[0001] The present invention relates generally to cell culture devices and, more particularly, to microfluidic cell culture devices and methods of making the same. Background Art

[0002] Organs-on-chip or microphysiological systems are a class of microfluidic platforms that cater to the need to recreate the physiologically relevant microenvironment of human or animal organs and can be used as in vitro models for disease research and drug development applications. The chip contains microchannels for guiding and manipulating tiny volumes of solutions, which can generally serve as effective simulations of human physiology and disease. These chips can be used to conduct experiments on biological cells and tissues in vitro. Organ chips allow for a higher level of control over the microenvironment that ensures tissue life support, as well as a means to directly observe cell and tissue behavior. Existing microfluidic organ chip designs include: i) synthetic membrane-based devices; ii) ECM-patterned or hydrogel-based platforms; iii) modular or directly connected microfluidic systems.

[0003] i) Synthetic membrane-based devices

[0004] A common approach to developing organ chips is to miniaturize the traditional transwell assay. The basic design consists of multiple layers of microchambers / fluidic channels that are juxtaposed and bonded to each other while being kept separated by a porous membrane. This configuration has been widely used to generate different organ chips, including the human intestine and lung. While unique shear stresses can be applied to the top and bottom chambers (on both sides of the porous membrane), any other actuation force cannot be discontinuous but must be applied to both chambers. Therefore, the flexibility of culturing two different cells / tissues (such as liver or kidney) that require different physiological biomechanics cannot be incorporated into the same device. Instead, multiple platforms will be required to achieve more complex biomimetic organ settings.

[0005] Multilayer devices have also been used to recreate other biomimetic cell culture platforms, notably blood vessels and the human heart. However, these platforms suffer from similar drawbacks as described above, in that only one cell or tissue type can be maintained in a platform, as the applied mechanical forces are constant across the entire platform. Another significant drawback of such membrane-based systems is the challenge of high-resolution microscopy, as the cells or tissues are located much larger than the working distance of conventional high-power objectives.

[0006] ii) ECM patterning or hydrogel-based platforms

[0007] Extracellular matrix (ECM) patterning is an alternative method that can be used to divide microchambers. This can be achieved by patterning ECM in defined channels within a polymer-based device or by creating an entire platform using ECM as a scaffold (hydrogel-based platform). Using this method, people have reconstructed a variety of organ chips including the intestine, liver, heart, blood vessels, and brain. These chips alleviate the problem of high-magnification imaging because they are usually single-layer devices, and the lateral arrangement of the channels allows the flexibility of merging multiple tissue chambers side by side laterally. However, current strategies for ECM patterned devices do not allow the use of active pumping sources such as syringe pumps to continuously perfuse liquids through these chambers for a long time (>24 hours). This is because active pumping has an adverse effect on the integrity of ECM hydrogels. In addition, due to the porous nature of these scaffolds, the manufacturing method of hydrogel-based platforms is cumbersome and time-consuming, and challenges are presented to fluid handling. In addition, in addition to the flow of fluid based on hydrostatic pressure and therefore the shear stress used for long-term culture, the current platform does not support the integration of any mechanical actuation.

[0008] iii) Modular or directly connected microfluidic systems

[0009] Another approach to generating organ chips is a modular microfluidic system approach, whereby different organ systems can be generated separately and then these different organ modules interconnected through external tubing or using microchannels or capillaries. While modular or compartmentalized microfluidic platforms do not face the problems faced by the above design considerations, especially in terms of imaging or the presence of ECM hydrogels, the use of convective fluidic channels to interconnect different organ chip channels introduces the complexity of maintaining different types of cells. This stems from the unique biochemical requirements of different organ systems.

[0010] Therefore, there is a need for an improved device that can provide greater design flexibility to easily modify and integrate multiple cell culture chambers for high-throughput organ-on-a-chip applications and also be able to simultaneously maintain different mechanical stimuli and / or biochemical environments in different cell culture chambers. Summary of the invention

[0011] According to a first aspect of the present disclosure, a microfluidic device is provided. The microfluidic device comprises: a substrate; a first layer disposed on the substrate, the first layer defining one or more cell culture chambers, each of the one or more cell culture chambers being configured to contain one or more cell cultures; and a second layer disposed on the first layer, the second layer defining one or more air chambers. Each of the one or more air chambers is adjacent to a corresponding cell culture chamber.

[0012] In certain embodiments, the base layer includes a transparent glass layer.

[0013] In certain embodiments, the first layer defines a plurality of cell culture chambers. The plurality of cell culture chambers are arranged in a lateral configuration.

[0014] In some embodiments, the second layer defines a corresponding plurality of air chambers. The device also includes a programmable pneumatic controller for operating the plurality of air chambers. The programmable pneumatic controller is configured to actuate the plurality of air chambers independently of one another.

[0015] In some embodiments, each of the plurality of air chambers is configured to generate a biomechanical cue in the corresponding cell culture chamber. The biomechanical cue includes at least one of fluid flow, shear stress, pressure gradient, or mechanical compression and expansion.

[0016] In certain embodiments, at least selected cell culture chambers of the plurality of cell culture chambers are connected to each other.

[0017] In certain embodiments, the first layer further defines a plurality of extracellular matrix (ECM) channels. Each ECM channel is adjacent to a respective one of the plurality of cell culture chambers.

[0018] In certain embodiments, the microfluidic device further comprises a capillary burst valve disposed between each of the plurality of ECM channels and a corresponding one of the plurality of cell culture chambers.

[0019] According to a second aspect of the present disclosure, a cell culture method is provided, which comprises: providing a microfluidic device as described above; providing one or more cell cultures in one or more cell culture chambers; and actuating one or more gas chambers so that each of the one or more gas chambers generates mechanical stimulation to the corresponding cell culture chamber.

[0020] In certain embodiments, providing one or more cell cultures in one or more cell culture chambers includes providing a cell culture of a first cell type in a first cell culture chamber and providing a cell culture of a second cell type different from the first cell type in a second cell culture chamber.

[0021] In certain embodiments, providing one or more cell cultures in one or more cell culture chambers further comprises providing one or more types of microorganisms along with the cell cultures in the cell culture chambers.

[0022] In certain embodiments, providing the one or more cell cultures in the one or more cell culture chambers further comprises controlling a flow of fluid in the one or more cell cultures through corresponding inlets and outlets of the one or more cell culture chambers.

[0023] In certain embodiments, actuating one or more air chambers includes actuating one or more air chambers independently of each other.

[0024] In certain embodiments, the method further comprises generating a mechanical stimulus in one or more cell culture chambers having one or more cell cultures by simultaneously actuating one or more gas chambers and controlling fluid flow through the one or more cell culture chambers.

[0025] In certain embodiments, the method further comprises controlling one or more cell cultures with one or more biochemical cues selected from the group consisting of proteins, peptides, oligonucleotides, chemicals, nanoparticles, or drugs.

[0026] In certain embodiments, controlling the one or more biochemical signals includes regulating oxygen concentration in the one or more cell culture chambers using an oxygen control module.

[0027] According to a third aspect of the present disclosure, a method for manufacturing a microfluidic device is provided. The method includes: providing a base layer; forming a first layer so that the first layer defines one or more cell culture chambers, each of the one or more cell culture chambers being configured to contain one or more cell cultures; forming a second layer so that the second layer defines one or more air chambers; disposing the first layer on the base layer; and disposing the second layer on the first layer so that each of the one or more air chambers is adjacent to a corresponding cell culture chamber.

[0028] In certain embodiments, forming the first layer includes spin coating polydimethylsiloxane (PDMS) on the first mold, and wherein forming the second layer includes casting the PDMS on the second mold.

[0029] In certain embodiments, disposing the first layer on the base layer comprises a first plasma bonding step, and wherein disposing the second layer on the first layer comprises a second plasma bonding step.

[0030] In certain embodiments, forming the first layer further comprises forming a plurality of extracellular matrix (ECM) channels, each ECM channel being adjacent to a respective one of the plurality of cell culture chambers.

[0031] Microfluidic devices according to the present disclosure can be used to construct a platform consisting of a microscope-grade glass cover slip and a multilayer chip with a built-in mechanical actuator. The platform allows the cultivation of single or multiple human-derived or animal-derived cells or their co-cultivation with microorganisms under static or dynamic culture conditions of fluid flow, shear stress, and cyclic mechanical compression and relaxation. Microfluidic devices provide the flexibility to adjust applications and reproduce multiple microphysiological systems (such as liver, intestines, blood vessels, kidneys, and heart). Multiple cell culture chambers with independently controllable biomechanical and biochemical signals contribute to drug or compound screening processes. In addition, the size or design of different cell culture chambers and mechanical actuators can be easily modified to adapt to different applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The specification sets forth a complete and enabling disclosure of the invention, including various embodiments and apparatus and methods for using the invention, for one of ordinary skill in the art, and the specification refers to the accompanying drawings, in which:

[0033] Figure 1 A microfluidic device according to one embodiment of the present disclosure is shown.

[0034] Figure 2 A cross-sectional view of a microfluidic device according to one embodiment of the present disclosure is shown.

[0035] Figure 3 Schematic diagrams showing the gas chamber and cell culture chamber in different states.

[0036] Figure 4 Depicted are graphs showing various states of a cell culture chamber in response to varying degrees of pneumatic actuation of the air chamber.

[0037] Figure 5A-5B Various designs of the second layer in a microfluidic device are shown.

[0038] Figure 6A-6B Various designs of the first layer in a microfluidic device are shown.

[0039] Figure 7 Microscopic images of a microfluidic device and a cell culture chamber according to one embodiment of the present disclosure are shown.

[0040] Figures 8A-8B A microfluidic device according to another embodiment of the present disclosure is shown.

[0041] Figures 9A-9B Microfluidic devices according to further embodiments of the present disclosure are shown.

[0042] Fig. 9C Microscopic images showing the diffusion of molecules through the ECM are shown.

[0043] Fig.9D Depicted is a graph showing fluorescence intensity over distance along the end portion of an ECM channel.

[0044] Fig.10 Shown is a microscopic image showing a three-culture system created by a microfluidic device according to one embodiment of the present disclosure.

[0045] Fig.11 Shown is a microscopic image showing bacterial colonization on a microfluidic device according to one embodiment of the present disclosure.

[0046] Fig.12 An example method of fabricating a microfluidic device according to one embodiment of the present disclosure is depicted.

[0047] Fig.13 A schematic diagram of the fabrication process is shown.

[0048] Fig.14 An example cell culture method according to one embodiment of the present disclosure is depicted. DETAILED DESCRIPTION

[0049] Reference will now be made in detail to embodiments of the invention, one or more examples of which are illustrated in the accompanying drawings. Each example is provided by way of explanation, not limitation, of the invention. Features shown or described as part of one embodiment may be used with another embodiment to produce a further embodiment. The disclosure is intended to cover such modifications and variations within the scope of the appended claims and their equivalents. The detailed description uses numerical and letter designations to refer to features in the drawings. The same or similar names in the drawings and description have been used to refer to the same or similar parts of the invention.

[0050] As used herein, the terms "first", "second" and "third" are used interchangeably to distinguish one component from another, and are not intended to indicate the position or importance of the various components. Unless the context clearly dictates otherwise, the singular forms "one", "an" and "the" include plural references. Unless otherwise specified herein, the terms "connect", "fix", "attach to", etc. refer to both direct connection, fixation or attachment, and indirect connection, fixation or attachment through one or more intermediate components or features. The terms "include", "comprise", "contain", "cover", "have", "have" or any other variant thereof used herein are intended to cover non-exclusive inclusion. For example, a process, method, article or device that includes a list of features is not necessarily limited to these features, but may include other features that are not explicitly listed or inherent to such processes, methods, articles or devices.

[0051] Approximate terms, such as "about," "approximately," "approximately," or "substantially," include values ​​that are greater than or less than 10% of the stated value.

[0052] Figure 1 A microfluidic device 10 according to one embodiment of the present disclosure is shown. The microfluidic device can be used as an organ chip and includes multiple layers with different functions. Figure 1 The microfluidic device 10 in the embodiment includes a base layer 100 , a first layer disposed on the base layer 200 , and a second layer 300 disposed on the first layer 200 .

[0053] The substrate 100 is configured to provide support for the other layers. The substrate 100 can be made of glass or other transparent materials. The transparent substrate enables optical analysis or observation of cells or tissues cultured in the first layer 200 from the bottom side of the device 10. In certain embodiments, the substrate 100 can be in the form of a glass coverslip, which makes the microfluidic device 10 easy to integrate with a high-resolution microscope to monitor and optically evaluate the cell or tissue culture process in real time. The thickness of the glass coverslip can be 80-300 μm, such as 130-170 μm, 160-190 μm, 190-250 μm. In one embodiment, the thickness of the glass coverslip can be about 150 μm, making the microfluidic device suitable for super-resolution and high-magnification (100x objective) imaging without the need for electron microscopy technology for analysis.

[0054] The first layer 200 is configured to provide a space for cell culture. The first layer 200 defines one or more cell culture chambers 210. Each chamber 210 can be configured to accommodate cells, micro-tissues and / or microorganisms grown and resident in a microfluidic device. Figure 1 In the illustrated embodiment, the first layer 200 defines an array of five cell culture chambers 210 having the same design. The cell culture chambers 210 can generally be arranged in a lateral configuration. In particular, the cell culture chambers 210 can be equally spaced along the longitudinal direction of the microfluidic device 10. In certain embodiments, the cell culture chambers 210 are isolated from each other so that different mechanical actuation modes can be applied to different cell culture chambers. In alternative variations, each cell culture chamber can have a unique design. It should be understood that the number of cell culture chambers and the size or shape of the cell culture chambers can be selected to suit the cells to be cultured.

[0055] The second layer 300 is used as an actuator layer to generate and regulate various mechanical forces on cells in the cell culture chamber in the first layer. The second layer 300 defines one or more air chambers 310. Actuation of the air chambers 310 enables the microfluidic device 10 to simulate dynamic physiological microenvironments, including, for example, heartbeat, air breathing, intestinal peristalsis, arteriolar contraction, etc. Similar to the first layer 200, Figure 1The second layer 300 shown defines an array of five air chambers 310 having the same design. The air chambers 310 can be equally spaced along the longitudinal direction of the microfluidic device. In certain embodiments, the air chambers 310 are isolated from each other so that the actuation of the air chambers can be independently controlled. In alternative variations, each air chamber can have a unique design. It should be understood that the number of air chambers and the size or shape of the air chambers can be selected according to the requirements of the specific application.

[0056] Figure 2 FIG. 1 is a cross-sectional view of a microfluidic device according to an embodiment of the present disclosure. A first layer 200 is supported on a base layer 100 . A second layer 300 is disposed on the first layer 200 . Figure 2 1 and 2. A cell culture chamber 210 and an air chamber 310 can be seen in the figure. In this embodiment, the cell culture chamber 210 is defined by the base layer 100 and the first layer 200. The cell culture chamber 210 may include a cavity or micropore formed in the first layer 200 and closed by the base layer 100. The base layer 100 prevents the cells or tissues in the cell culture chamber from being exposed to the atmosphere. The air chamber 310 is defined by the first layer 200 and the second layer 300. The air chamber 310 may include a micropore formed in the second layer 300 and closed by the first layer 200.

[0057] Figure 1 The microfluidic device 10 in the embodiment includes five cell culture chambers 210 and five air chambers 310. Each of the plurality of air chambers 310 in the second layer 300 is located adjacent to a corresponding cell culture chamber 210, so that the movement of the air chambers 310 in the second layer 300 changes the microenvironment in the corresponding cell culture chamber 210 in the first layer 200. Figure 2 As shown, the cell culture chamber 210 is vertically aligned with the air chamber 310. A portion of the upper portion of the first layer 200 forming a roof 201 of the cell culture chamber 210 separates the cell culture chamber 210 from the air chamber 310. It should be understood that the two chambers may be separated by a partition member of other designs.

[0058] The first layer 200 and the second layer 300 formed with one or more chambers 210, 300 may be manufactured using techniques such as injection molding, compression injection molding, embossing, photolithographic etching, laser etching, and / or other methods. Figure 1 Each of the first layer 200 and the second layer 300 in the illustrated microfluidic device 10 is a single integral body. In alternative variations, the first layer and the second layer may have discrete portions that are coupled together.

[0059] Pneumatic actuation may be used to expand or contract the air chamber 310. Figure 3As shown, a programmable pneumatic controller can be used to control the percentage of expansion and contraction. The air chamber 310 acts as an actuator to generate a large amount of mechanical force on the cells or tissues in the cell culture chamber. The mechanical force can be controlled to cause periodic relaxation and compression of the cell culture chamber, and / or simulate peristaltic fluid dynamics. Therefore, the air chamber 310 generates different mechanical stimuli in the cell culture chamber 210. On the other hand, the inlet and outlet of the cell culture chamber 210 allow the cell culture medium or appropriate agent (e.g., drug) to be introduced to be perfused into the cell culture chamber 210 under constant perfusion controlled by a fluid handling pump (e.g., a syringe pump, a pressure pump, or a peristaltic pump). Perfusion culture produces shear stress, which is a basic biomechanical signal for cell growth.

[0060] Figure 4 A graph showing various states of a cell culture chamber in response to varying degrees of pneumatic actuation of an air chamber is depicted. The four curves in the graph are 3D reconstructions of microscopic images of a cell culture chamber top plate in different states during a relaxation and compression cycle. The 0% curve is generally linear, corresponding to a state in which the cell culture chamber undergoes no relaxation or compression. The 30% curve is a convex curve, corresponding to a state in which the cell culture chamber undergoes moderate relaxation in response to contraction of the air chamber. The 60% curve is a concave curve, corresponding to a state in which the cell culture chamber undergoes moderate compression in response to a moderate degree of expansion of the air chamber. The 100% curve is a concave curve, corresponding to a state in which the cell culture chamber undergoes full compression in response to a high degree of expansion of the air chamber.

[0061] Figure 5A-5B Various designs of the second layer in the microfluidic device are shown. The second layer serves as an actuator layer and may include one or more air chambers. The actuator layer shown in view (a) includes five air chambers 310 arranged along the longitudinal direction of the actuator layer. Each air chamber 310 has an air inlet 311, an air cavity 312, and an air passage 313 connecting the air inlet 311 and the air cavity 312. The air passage 313 extends in a direction perpendicular to the air cavity 312. The width of the air passage 313 can be less than the diameter of the air inlet 311 to limit the air flow rate.

[0062] Each air chamber can be configured to generate a biomechanical signal in the corresponding cell culture chamber. The biomechanical signal can include at least one of fluid flow, shear stress, pressure gradient, or mechanical compression and expansion. For example, air can be introduced from the air inlet 311 into the air cavity 312 to expand the air chamber 310, which in turn compresses the cell culture chamber 210 below. Removing air from the air cavity causes the air chamber to shrink, thereby relaxing the cell culture chamber.

[0063] Each actuator layer shown in views (b) and (c) has a single air chamber. The air inlet 311 can be arranged on one side of the air cavity 312. The air cavity 312 can have an elongated shape, such as a rectangle or an ellipse. The aspect ratio of the rectangular air cavity can be greater than 1.5, for example in the range of 2 to 5. The air inlet 311 can have a circular cross-section. In an alternative variation, each air chamber can have multiple air inlets and multiple air channels. One or more air inlets can be controlled with a programmable pneumatic controller. In some embodiments, the pneumatic controller is configured or programmed to actuate multiple air chambers independently of each other.

[0064] Each actuator layer shown in views (d) and (e) has an array of three air chambers 310. The air chambers 310 can be arranged in a more compact manner compared to the design in view (a). The air cavity 312 can have a square or circular shape.

[0065] Each actuator layer shown in views (f) to (i) includes a single air inlet 311 and a plurality of air cavities 312. In views (f), (h) and (i), a single air inlet 311 is connected to each air cavity 312 via an air channel network 314. In view (g), no direct fluid communication is provided between the intermediate air cavity 312 and the air inlet 311. It should be understood that the air channel network (e.g., number of branches, length, width, direction, etc.) can be designed according to the specific application.

[0066] Figure 6A-6B Various designs of the first layer in a microfluidic device are shown. The first layer is used as a cell culture layer and may include one or more cell culture chambers. The cell culture layer in view (a) has a single cell culture chamber 210. The cell culture chamber 210 includes a cell chamber 211, an inlet 212, an outlet 213, an inlet channel 214 connecting the cell chamber 211 and the inlet 212, and an outlet channel 215 connecting the cell chamber 211 and the outlet 213. Cell culture medium or agent can flow through the cell culture chamber 211 via the inlet 212 and the outlet 213. The cell chamber 211 may have an elongated shape as shown in view (a). Each of the inlet channel 214 and the outlet channel 215 may form an angle with the cell chamber 211. The angle may be in the range of 90 to 180 degrees, for example, 100 to 150 degrees, 110 to 140 degrees, or 115 to 130 degrees. The obtuse angle avoids a sudden change in direction from the inlet 212 to the outlet 213.

[0067] The design of the cell culture chamber can vary depending on the specific application. In some embodiments, the shape of the cell chamber matches the shape of the air cavity. In alternative embodiments, a cell cavity can be affected by a group of air cavities. When viewed from above, each air cavity in the group only covers a portion of the cell cavity.

[0068] Compared to view (a), the cell culture layer in view (b) further includes a microchannel 220. The microchannel 220 extends from the inlet 221 and is adjacent to the cell culture chamber 210. In certain embodiments, the microchannel 220 may be oriented parallel to the cell cavity 211. The microchannel 220 may be adjacent to the cell cavity 211 over a portion of its length (e.g., over 1 / 5 of its length, over 1 / 3 of its length, or over half of its length).

[0069] Microchannel 220 can be an extracellular matrix (ECM) channel. The ECM channel is used to accommodate an ECM hydrogel or ECM hydrogel combination, such as type I collagen, type IV collagen, matrix glue (Matrigel), fibrinogen, hyaluronic acid hydrogel, synthetic hydrogel, such as gelatin-based (GelMA) or polyethylene glycol (PEG)-based hydrogel. Such hydrogels are very important for long-term cell and tissue maintenance.

[0070] In one embodiment, the ECM channel leads to the cell culture chamber through a capillary burst valve (CBV), which ensures that the ECM hydrogel loaded into the ECM channel does not overflow into the cell culture chamber. The capillary burst valve can be integrated into the microfluidic device design to allow the incorporation of multiple interconnected parallel channels without the need for external tubing or fittings. However, it should be noted that the capillary burst valve is not a necessary element of the present invention and can be omitted or removed. Other types of flow control devices can also be used to regulate the flow of the ECM hydrogel into the cell culture chamber.

[0071] Each cell culture layer shown in views (c), (d) and (e) includes a plurality of cell culture chambers 210 and ECM channels 220. In view (c), two cell culture chambers 210 are arranged so that two cell cavities 211 face each other. The ECM channel 220 is sandwiched between the two cell cavities 211. The ECM hydrogel can flow into the two cell cavities 211 through the ECM channel 220. A capillary burst valve can be provided between each side of the ECM channel and the corresponding cell culture chamber. In view (d), the ECM channel 220 is bifurcated. Each branch 222 of the ECM channel 220 is adjacent to a cell cavity 211. A capillary burst valve can be provided between each branch and the corresponding cell culture chamber. The cell culture layer in view (e) includes three cell culture chambers and an ECM channel 220 having three branches 222. Each branch leads to a corresponding cell culture chamber. A capillary burst valve can be provided at the end portion of each branch to control the flow of the ECM hydrogel.

[0072] Each cell culture layer shown in views (f), (g) and (h) includes an array of cell culture chambers. The design in view (f) has four cell culture chambers and can be considered as a combination of the two units shown in view (d). The ECM channel is configured as a channel network 230 having a single inlet 231 and four branches 232. Each branch 232 is adjacent to a cell culture chamber 210. Similarly, the cell culture layer in view (f) is a combination of the three units shown in view (d). It should be understood that the shape, number and arrangement of the cell culture chambers in the cell culture layer can be changed to accommodate different applications. If a large number of cell culture chambers are used, the ECM channel network 230 may have a complex design. Alternatively, the cell culture layer may include a plurality of ECM channels 220 separated from each other, such as the embodiment shown in view (h). The separate ECM channels 220 allow different ECM hydrogels to enter different cell culture chambers 210.

[0073] The actuator layer and the cell culture layer can be made of polymers, such as polydimethylsiloxane (PDMS), polystyrene (PS), polycarbonate (PC) and / or polysiloxane. The materials of the actuator layer and the cell culture layer can have any degree of transparency, reflectivity or other optical properties. For example, the material can be transparent so that optical analysis can be performed. Additionally or alternatively, a portion of the layer can be opaque, translucent or have any suitable opacity.

[0074] The design of the cell culture layer can be changed for different applications. In addition, depending on the configuration of the air chambers in the actuator layer, the mechanical actuation applied to each cell culture chamber can be individually controlled. The microfluidic device according to the present disclosure can be used to construct a high-throughput cell culture system that is capable of maintaining cells or tissues over long periods of time and also maintaining the required mechanical actuation and perfusion culture. Four example applications of the microfluidic device are described below.

[0075] Example 1: Microfluidic Device with Parallel Cell Culture Chambers

[0076] Figure 1 The microfluidic device 10 shown in FIG. 1 was used to develop a biomimetic intestinal epithelium using the Caco-2 cell line, which is widely used as a model of the intestinal epithelial barrier. Figure 7Microscopic images of microfluidic devices and cell culture chambers are shown. Hydrogel is introduced into ECM channel 220 from ECM inlet 221. Protein coating solution including a combination of one or more proteins such as type I collagen, fibronectin, gelatin or matrigel is introduced into cell culture chamber 210 through ECM channel 220 to promote cell attachment and culture. Caco-2 cells are then introduced from cell chamber inlet 212 and adhered to a glass substrate. Culture is maintained for 6-8 days under the influence of continuous perfusion and periodic compression and relaxation. As described above, the periodic compression and relaxation of each cell culture chamber 210 can be achieved by independently controlling the air introduced and removed from air chamber 310 through air inlet 311. Immunostaining is then performed and high-resolution images of Caco-2 cells are obtained by fluorescence microscopy.

[0077] Although the test was performed using a microfluidic device with five parallel arranged cell culture chambers, it should be understood that the microfluidic device according to the present disclosure can be expanded to include a greater number of cell culture chambers. The arrangement of the cell culture chambers can be modified to include multiple rows and columns.

[0078] Example 2: Microfluidic Device with Interconnected Cell Culture Chambers

[0079] Figures 8A-8B 1 shows a microfluidic device according to an alternative embodiment of the present disclosure. The microfluidic device has a glass layer 100, a cell culture layer 200 disposed on the glass layer 100, and an actuator layer 300 disposed on the cell culture layer 200. The cell culture layer may be formed by Fig. 6A The design shown in view (e) in FIG. 2 . The ECM channel 220 formed in the cell culture layer 200 includes an ECM inlet 221 and three branches 222, which are connected to three cell culture chambers 241, 242, and 243, respectively. In this example, the ECM channel is configured as a device for interconnecting multiple cell culture chambers. Since the ECM is porous, it allows liquid to diffuse through it while preventing any convection flow.

[0080] Although the three cell culture chambers have the same design, their associated gas chamber patterns can be different. Figure 8B As best shown in FIG. 1 , the cell culture chamber 241 on the left is affected by an air chamber 321 composed of two smaller air chambers 331, each of which covers only a portion of the cell culture chamber 241. The air chamber design is similar to Figure 5B The design shown in the view (f) of FIG. The cell culture chamber 242 on the right is affected by the air chamber 322 having a larger air cavity 332. No air chamber is provided for the cell culture chamber 243 at the bottom. Therefore, unique mechanical stimulation can be generated in different cell culture chambers.

[0081] Figures 9A-9B2 shows a microfluidic device according to another embodiment of the present disclosure. The actuator layer 300 is similar to Figures 8A-8B The cell culture layer 200 includes two cell culture chambers 210 facing each other and connected to each other through an ECM channel 240. The ECM channel 240 includes a main portion 241 and an end portion 242, and the end portion 242 extends between the two cell culture chambers 210 in a direction substantially perpendicular to the main portion 241. The opposite end 243 of the end portion 242 leads to the two cell culture chambers 210.

[0082] Small molecule diffusion can be observed and studied using fluorescence microscopy. Fig. 9C Microscopic images showing the diffusion of 4.4 kDa FITC-dextran molecules are shown to study its diffusion profile through the ECM. Fig.9D Plotted to show the distance a-a' along the terminal segment of the ECM channel (in Fig. 9C Graph of fluorescence intensity on ).

[0083] Example 3: Co-culturing different cell types using a microfluidic device

[0084] In addition to providing unique mechanical actuation in the cell culture chamber, the microfluidic device according to the present disclosure can also be used to co-culture different cell types belonging to different human organs. In this example, the microfluidic device is used to culture endothelial tubes using human microvascular endothelial cells in one chamber and Caco-2 cells in an adjacent chamber. Different mechanical forces can be applied to the cells. The culture is maintained for 7 days. In addition to adherent cells, suspended cells (such as immune cells) can also be maintained in the cell culture chamber. Therefore, this example shows the ability of a microfluidic device to create a three-culture system involving three different cell lines, which makes it possible to produce a multi-organ chip model.

[0085] Fig.10 Microscopic images showing a tri-culture system created by a microfluidic device are shown. Human microvascular endothelial cells and human colon adenocarcinoma cells, which resemble intestinal epithelial cells, were cultured on either side of the ECM and maintained under unique mechanical actuation. The arrow in the cell compartment 1 image indicates a suspended human monocytic cell line (THP-1) cultured within the endothelial cell tube. Fig.10 The inset in shows cell growth in the corresponding cell compartment under mechanical actuation.

[0086] Example 4: Co-culturing microorganisms using a microfluidic device

[0087] Additional applications of microfluidic devices according to the present disclosure include co-culture of microorganisms with mammalian cells. Fluid flow, shear stress, and mechanical actuation that mimics dynamic organ systems enable relatively long-term co-culture (>48 hours) of mammalian cells with one or more types of microorganisms, which is challenging in conventional cell culture systems because of the rapid growth rate of microorganisms compared to mammalian cells.

[0088] In this example, a microfluidic device was used to demonstrate the co-culture of Caco-2 cells, mimicking intestinal epithelial cells, and various gut microbes, such as Escherichia coli and Lactobacillus rhamnosus. Caco-2 cells were cultured under different mechanical forces for 7 days, and on day 6, gut microbes were introduced and co-cultured for up to 48 hours.

[0089] Fig.11 Microscopic images showing bacterial colonization on a microfluidic device are shown. Images show: (A) colonization of GFP-E. coli on a Caco-2 cell mucus layer; (B) colonization of GFP-E. coli on recreated microvilli; and (C) colonization of Lactobacillus rhamnosus on recreated microvilli. This example shows the possibility of recreating a host-microbe model system using a microfluidic device.

[0090] Fig.12 An example method 400 of fabricating a microfluidic device according to one embodiment of the present disclosure is depicted. Fig.13 A schematic diagram of the manufacturing process is shown. In one or more embodiments, the steps may be omitted, repeated, and / or performed in a different order. Fig.12 and 13 Therefore, embodiments of the manufacturing method should not be considered limited to the specific arrangements of elements shown in the figures.

[0091] In block 410, a base layer is provided. Base layer 100 ( Fig.13 ) can be made of transparent material, which enables optical analysis of cells or tissues cultured in the microfluidic device. In some embodiments, the substrate can be a cover glass, preferably a microscope grade glass cover glass, so that the microfluidic device is compatible with super-resolution microscopy.

[0092] In block 420, a first layer is formed. The first layer 200 ( Fig.13 ) is used as a cell culture layer. The first layer 200 defines one or more cell culture chambers 210 ( Fig.13 ). Each of the one or more cell culture chambers 210 is configured to accommodate cells, tissues, or microorganisms. In certain embodiments, as Fig.13 As shown, the first layer 200 (bottom layer) may be formed by spin coating a polymer on a first mold 421. The first mold 421 may be a silicon mold fabricated using SU-8 based photolithography technology.

[0093] In certain embodiments, forming the first layer may include forming a plurality of extracellular matrix (ECM) channels in the first layer. Each ECM channel is adjacent to a cell culture chamber to allow introduction of an ECM hydrogel or a combination of ECM hydrogels into the cell culture chamber.

[0094] In block 430, a second layer is formed. The second layer 300 ( Fig.13 ) is used as an actuator layer. The second layer 300 defines one or more air chambers 310. In some embodiments, such as Fig.13 As shown, the second layer 300 (top layer) may be formed by casting a polymer on the second mold 431. Also, the second mold 431 may be a silicon mold fabricated using SU-8 based photolithography technology.

[0095] In certain embodiments, both the first layer 200 and the second layer 300 are made of polydimethylsiloxane (PDMS). To form the first layer 200, PDMS (preferably in a ratio between 10:1 and 15:1 (w / w)) is spin-coated on a silicon mold to produce a PDMS thickness of less than 1 mm (e.g., less than 500 μm) for accommodating the cell culture chamber. To form the second layer 300, a thicker PDMS layer (preferably in the range of 2 mm to 3 mm) is cast on another silicon mold to create the desired air chamber configuration.

[0096] In certain embodiments, access ports (e.g., inlets and outlets) are formed on the first layer and the second layer. The ports on the first layer can be used to direct liquid or gas into and out of the cell culture chamber. The ports on the second layer can be used to introduce air into and remove air from the air chamber.

[0097] In block 440, a first layer is disposed on the base layer. Fig.13 As shown, a first stage plasma bonding process may be performed to attach a first layer 200 (eg, a thinner PDMS layer) to a base layer 100 (eg, a glass coverslip).

[0098] Finally, in block 450, a second layer is disposed on the first layer. Proper alignment between the air chambers in the second layer and the cell culture chambers in the first layer may be required to ensure that each air chamber is adjacent to a corresponding cell culture chamber. Fig.13 As shown, a second stage plasma bonding process may be performed to attach the second layer 300 (eg, a thicker PDMS layer) to the first layer 200 (eg, a thinner PDMS layer).

[0099] The microfluidic device fabricated using the above method has a multilayer configuration, including a glass cover slip at the bottom, a PDMS cell culture layer in the middle, and a PDMS actuator layer at the top. By adjusting the design of the actuator layer, unique mechanical actuation patterns can be applied to individual cell culture chambers in the cell culture layer. The microfluidic device according to the present disclosure represents a significant improvement over commercially available platforms, in which the same mechanical actuation must be applied to all cell culture chambers.

[0100] Fig.14 An exemplary cell culture method 500 according to one embodiment of the present disclosure is depicted. For example, the above reference Figure 1-10 Describe the microfluidic device to practice Fig.14 The method in .

[0101] First, in block 510, a microfluidic device is provided. The microfluidic device may be a multilayer organ chip, including a substrate, a cell culture layer supported on the substrate, and an actuator layer disposed on the first layer. Figure 12-13 Methods to fabricate microfluidic devices are described.

[0102] The cell culture layer may define one or more cell culture chambers. The cell culture layer may have Figure 6A-6B The actuator layer may define one or more air chambers. The actuator layer may have a configuration according to Figure 5A-5B The layout of any embodiment or combination of embodiments shown in .

[0103] In block 520, one or more cells are provided in one or more cell culture chambers. In certain embodiments, a first cell type may be cultured in a first cell culture chamber. A second cell type different from the first cell type may be cultured in a second cell culture chamber. The first and second cell culture chambers may share the same design or may have different patterns. Thus, different types of cells may be cultured simultaneously using a single microfluidic device.

[0104] In certain embodiments, one or more types of microorganisms may be provided in the cell culture chamber together with the cells. The above Example 4 shows that the microfluidic device according to the present disclosure allows for co-culturing of microorganisms and cells.

[0105] In certain embodiments, fluid flow in one or more cell cultures may be controlled through corresponding inlets and outlets of one or more cell culture chambers.

[0106] In block 530, one or more air chambers in the actuator layer are actuated. The actuation causes the air chambers to expand or contract, thereby generating mechanical stimulation in the corresponding cell culture chambers. In some embodiments, one or more air chambers can be actuated independently of each other, such as by using a programmable controller. Thus, biomechanical signals for cell growth in different cell culture chambers can be independently controlled. Biomechanical signals can include fluid flow, shear stress, pressure gradients, and / or mechanical compression and expansion.

[0107] In certain embodiments, mechanical stimulation may be generated by simultaneously actuating one or more gas chambers and controlling fluid flow through one or more cell culture chambers.

[0108] In block 540, one or more biochemical signals in one or more cell culture chambers are controlled. The biochemical signal may include a protein, a peptide, an oligonucleotide, a chemical substance, a nanoparticle, or a drug. In certain embodiments, controlling the biochemical signal may include adjusting the oxygen concentration in the one or more cell culture chambers using an oxygen control module. Thus, the method significantly shortens the time required to perform a comparative study of cell growth under different biochemical signals.

[0109] The present disclosure provides a multi-layer, multi-chamber platform with or without an integrated mechanical actuator. The integrated mechanical actuator may include multiple air chambers that enable different mechanical forces (e.g., fluid flow, shear stress, pressure gradient, mechanical compression, and expansion) to be maintained simultaneously in different cell culture chambers. The platform may define an array of cell culture chambers to enable high-throughput drug or compound screening. The multi-layer structure allows the user to easily and comfortably modify the size or design of the cell culture chambers and mechanical actuators according to the requirements of different applications.

[0110] Furthermore, capillary burst valves can be integrated into the platform to enable the incorporation of multiple interconnected parallel cell culture chambers without the need for external tubing or fittings. Furthermore, the use of thin glass coverslips as a substrate makes the platform suitable for super-resolution microscopy and compatible with high-magnification objectives with short working distances.

[0111] The various examples above show that the platform can be used, for example, to create an immune-competent vascularized human intestinal model by adding relevant cell types and culturing various microorganisms. This helps develop a human intestinal microphysiological system with relevant human intestinal cells (primary cells or cell lines), microorganisms, endothelial cells, and immune cells. This microphysiological system can be used to study host-microorganism interactions, microorganism-induced disease modeling, and drug development.

[0112] The platform also allows for long-term pump-free or pump-based perfusion for the culture of mammalian cells, microorganisms, or both. Thus, the platform provides users with the flexibility to tailor applications and recreate a variety of microphysiological systems (e.g., liver, intestine, vascular, kidney, and heart).

[0113] This description uses examples to disclose the invention and to enable those skilled in the art to practice the invention, including making and using any device or system and performing any included method. The patentable scope of the invention is defined by the claims and may include other embodiments that occur to those skilled in the art. These other embodiments are intended to be within the scope of the claims if they include elements that do not differ from the literal language of the claims, or if they include equivalent elements that do not differ substantially from the literal language of the claims.

Claims

1. A microfluidic device comprising: Grassroots; a first layer disposed on the base layer, the first layer defining one or more cell culture chambers, each of the one or more cell culture chambers being configured to house one or more cell cultures; as well as a second layer disposed on the first layer, the second layer defining one or more air chambers, Wherein, each of the one or more air chambers is adjacent to a corresponding cell culture chamber.

2. The device according to claim 1, wherein: The base layer includes a transparent glass layer.

3. The device according to claim 1 or 2, wherein: The first layer defines a plurality of cell culture chambers, and wherein the plurality of cell culture chambers are arranged in a lateral configuration.

4. The device according to claim 3, wherein: The second layer defines a corresponding plurality of air chambers, and wherein the apparatus further comprises a programmable pneumatic controller for operating the plurality of air chambers, the programmable pneumatic controller being configured to actuate the plurality of air chambers independently of one another.

5. The device according to claim 4, wherein: Each of the plurality of air chambers is configured to generate a biomechanical signal in a corresponding cell culture chamber, the biomechanical signal comprising at least one of fluid flow, shear stress, pressure gradient, or mechanical compression and expansion.

6. The device according to claim 4 or 5, wherein: At least selected cell culture chambers among the plurality of cell culture chambers are connected to each other.

7. The device according to claim 6, wherein: The first layer also defines a plurality of extracellular matrix (ECM) channels, each ECM channel being adjacent to a respective one of the plurality of cell culture chambers.

8. The device of claim 7, further comprising a capillary burst valve disposed between each of the plurality of ECM channels and a corresponding one of the plurality of cell culture members.

9. A cell culture method comprising: Providing a microfluidic device as defined in any one of claims 1 to 8; providing one or more cell cultures in the one or more cell culture chambers; and The one or more air chambers are actuated so that each of the one or more air chambers generates a mechanical stimulus to a corresponding cell culture chamber.

10. The method according to claim 9, wherein: Providing one or more cell cultures in the one or more cell culture chambers includes providing a cell culture of a first cell type in a first cell culture chamber and providing a cell culture of a second cell type different from the first cell type in a second cell culture chamber.

11. The method according to claim 9 or 10, wherein: Providing one or more cell cultures in the one or more cell culture chambers also includes providing one or more types of microorganisms in the cell culture chambers along with the cell cultures.

12. The method according to any one of claims 9 to 11, wherein: Providing one or more cell cultures in the one or more cell culture chambers further comprises controlling fluid flow in the one or more cell cultures through respective inlets and outlets of the one or more cell culture chambers.

13. The method according to any one of claims 9 to 12, wherein: Actuating the one or more air chambers includes actuating the one or more air chambers independently of each other.

14. The method of claim 12, comprising generating mechanical stimulation in one or more cell culture chambers having one or more cell cultures by simultaneously actuating the one or more gas chambers and controlling fluid flow through the one or more cell culture chambers.

15. The method according to any one of claims 9 to 14, further comprising controlling one or more biochemical signals of the one or more cell cultures, the one or more biochemical signals being selected from the group consisting of proteins, peptides, oligonucleotides, chemicals, nanoparticles or drugs.

16. The method according to claim 15, wherein: Controlling the one or more biochemical signals includes regulating oxygen concentration in the one or more cell culture chambers using an oxygen control module.

17. A method of manufacturing a microfluidic device, the method comprising: Provide grass-roots; forming a first layer such that the first layer defines one or more cell culture chambers, each of the one or more cell culture chambers being configured to house one or more cell cultures; forming a second layer such that the second layer defines one or more air cells, disposing the first layer on the base layer; and The second layer is disposed on the first layer such that each of the one or more air chambers is adjacent to a corresponding cell culture chamber.

18. The method according to claim 17, wherein: Forming the first layer includes spin coating polydimethylsiloxane (PDMS) on a first mold, and wherein forming the second layer includes casting PDMS on a second mold.

19. The method according to claim 17 or 18, wherein: Disposing the first layer on the base layer comprises a first plasma bonding step, and wherein disposing the second layer on the first layer comprises a second plasma bonding step.

20. The method according to any one of claims 17 to 19, wherein: Forming the first layer further includes forming a plurality of extracellular matrix (ECM) channels, each ECM channel being adjacent to a respective one of the plurality of cell culture chambers.

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