In-situ monitoring method, organ chip, manufacturing method and application

Abstract

The invention provides an in-situ monitoring method, an organ chip, a manufacturing method and application, and the in-situ monitoring method of the organ chip comprises the following steps: a culture membrane of the organ chip receives cells, and the culture membrane is integrated with a stretchable strain electrode; the cells are cultured in the culture chambers on the two sides of the culture membrane; controlling the pressure of the culture chamber to change so as to actuate the culture membrane; and electric signal output of the strain electrode is collected so as to monitor the strain of the culture membrane. By using the culture membrane with the strain electrode, the strain electrode can more truly and accurately collect strain data of the culture membrane, so that the organ chip can more accurately monitor the strain of the culture membrane. The invention relates to the field of organ chips.

Description

Technical Field

[0001] This application relates to the field of organ-on-a-chip, and in particular to an in-situ monitoring method, organ-on-a-chip, fabrication method and application. Background Technology

[0002] Organ-on-a-chip technology is a microfluidic chip used to simulate tissues and organs in vitro. Compared to traditional two-dimensional cell culture and animal models, organ-on-a-chip technology can more realistically simulate the pathological and toxicological interactions between different organs or tissues, reflecting the synergistic response of multiple organs to drugs.

[0003] Continuous monitoring of key quality parameters of organ-on-a-chip systems using label-free, non-destructive, reliable, high-throughput, and multiple methods is crucial for assessing the condition of organ-on-a-chip systems. In-situ real-time strain monitoring is particularly important, for example, monitoring key mechanical characteristics of cardiomyocyte contraction / relaxation processes in the heart (e.g., beat frequency, intensity, and regularity of beat patterns), monitoring alveolar cell respiratory rate and intensity in alveolar-on-a-chip systems, and monitoring muscle tissue.

[0004] In related technologies, Olivier T. Guenat et al. constructed a microimpedance computed tomography system, which can monitor mechanical changes occurring in the alveolar barrier using impedance coplanar electrodes, achieving in-situ monitoring of strain. However, this method is limited to specific organ-on-a-chip structures and lacks transferability. Furthermore, this method indirectly reflects the strain of the culture membrane by combining changes in solution impedance with microscopic measurements, which suffers from insufficient accuracy. Summary of the Invention

[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes an in-situ monitoring method, an organ-on-a-chip, a fabrication method, and an application. The in-situ monitoring method can more accurately monitor the strain of the culture membrane.

[0006] The in-situ monitoring method for organ-on-a-chip provided in this application is characterized by comprising: receiving cells in a culture membrane of the organ-on-a-chip, wherein the culture membrane integrates a stretchable strain electrode; culturing the cells in culture chambers on both sides of the culture membrane; controlling the pressure of the culture chambers to change, thereby actuating the culture membrane; and acquiring the electrical signal output of the strain electrode to monitor the strain of the culture membrane.

[0007] According to the in-situ monitoring method provided in this application, at least the following technical effects are achieved: by using a culture membrane with strain electrodes, the strain electrodes can collect strain data of the culture membrane more realistically and accurately, thereby enabling organ-on-a-chip to monitor the strain of the culture membrane more accurately.

[0008] According to some embodiments of this application, the in-situ monitoring method further includes calibrating the stress-strain relationship of the culture membrane before receiving the cells.

[0009] According to some embodiments of this application, the strain electrode is transparent.

[0010] The organ-on-a-chip according to this application includes a first slice, a second slice, and a culture membrane. The first slice forms a first chamber, the second slice forms a second chamber, and the culture membrane is located between the first slice and the second slice, separating the first chamber and the second chamber. The culture membrane includes a substrate and a strain electrode, the strain electrode being disposed on the substrate, and the substrate and the strain electrode being capable of stretching and deformation.

[0011] According to the organ-on-a-chip provided in this application, it has at least the following technical effects: by using strain electrodes that can be stretched and deformed, and then integrating the strain electrodes on the culture membrane, the strain electrodes can collect strain data of the culture membrane more realistically and accurately, so that the organ-on-a-chip can monitor the strain of the culture membrane more accurately.

[0012] According to some embodiments of this application, the strain electrode includes a deformation portion located within the first chamber.

[0013] According to some embodiments of this application, the deviation between the stiffness of the deformed portion and the stiffness of the substrate does not exceed ±5%.

[0014] According to some embodiments of this application, the culture membrane is light-transmitting.

[0015] According to some embodiments of this application, the strain electrode has a porous structure.

[0016] According to some embodiments of this application, the substrate is made of a transparent material.

[0017] According to some embodiments of this application, the culture membrane further includes a signal line connected to the strain electrode, the signal line extending out of the substrate, and the signal line being used to connect to an external terminal.

[0018] According to some embodiments of this application, the portion where the signal line connects to the strain electrode is covered with a protective layer.

[0019] According to some embodiments of this application, the material of the strain electrode includes one or more of graphene, carbon nanotubes, carbon fibers, and MXene.

[0020] According to some embodiments of this application, the substrate material includes one or more of PDMS, dual-network hydrogel, and elastic gel.

[0021] According to some embodiments of this application, the first chamber is used for culturing cells, and the second chamber is used for actuating the culture membrane.

[0022] According to some embodiments of this application, the organ-on-a-chip includes an inlet channel and an outlet channel. The inlet channel is connected to the first chamber and is used to input culture medium. The outlet channel is connected to the first chamber and is used to collect waste liquid.

[0023] According to some embodiments of this application, the organ-on-a-chip includes a gas channel communicating with a second chamber, the gas channel being used to regulate the gas pressure in the second chamber.

[0024] According to some embodiments of this application, the strain electrode is located on the side of the culture membrane closer to the second chamber.

[0025] According to some embodiments of this application, the organ-on-a-chip further includes a third sheet and an actuation membrane, the third sheet forming a third chamber, the actuation membrane being located between the second sheet and the third sheet, and the actuation membrane separating the second chamber and the third chamber.

[0026] The method for fabricating a culture membrane according to this application includes: forming a strain electrode on a carrier; forming a substrate on the carrier and bonding it with the strain electrode; and removing the carrier.

[0027] According to some embodiments of this application, the method for fabricating the culture membrane further includes forming a signal line connected to the strain electrode.

[0028] According to some embodiments of this application, the method for fabricating the culture membrane further includes forming a protective layer covering the connection portion of the signal line and the strain electrode.

[0029] According to some embodiments of this application, forming the strain electrode on the carrier includes forming laser-induced graphene on a PI film by laser reduction.

[0030] According to some embodiments of this application, forming a substrate on the carrier and bonded to the strain electrode includes spin-coating a PDMS solution on the carrier and curing the PDMS solution.

[0031] The application of organ-on-a-chip in in situ monitoring according to this application.

[0032] The culture membrane fabrication method provided in this application includes the organ-on-a-chip provided in this application. Therefore, the culture membrane fabrication method has the beneficial effects provided by the organ-on-a-chip, which will not be elaborated here. Attached Figure Description

[0033] The accompanying drawings are used to provide a further understanding of the technical solutions disclosed in this application and form part of the specification. They are used together with the embodiments disclosed in this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions disclosed in this application.

[0034] Figure 1 This is a schematic diagram of the structure of an organ-on-a-chip according to an embodiment of the first aspect of this application;

[0035] Figure 2 This is an exploded view of an organ-on-a-chip according to an embodiment of the first aspect of this application;

[0036] Figure 3 This is a cross-sectional view of an organ-on-a-chip according to an embodiment of the first aspect of this application;

[0037] Figure 4 This is a schematic diagram of the structure of the culture membrane according to an embodiment of the first aspect of this application;

[0038] Figure 5 This is a schematic diagram of the macroscopic pore structure of the culture membrane according to an embodiment of the first aspect of this application;

[0039] Figure 6 This is a schematic diagram of the structure of an organ-on-a-chip according to an embodiment of the second aspect of this application;

[0040] Figure 7 These are experimental data based on the in-situ monitoring method of this application, which show the correlation between pressure and strain;

[0041] Figure 8 These are experimental data based on the in-situ monitoring method of this application, which show the correlation between pressure and resistance;

[0042] Figure 9 These are experimental data based on the in-situ monitoring method of this application, showing the results of strain monitoring.

[0043] Figure label:

[0044] First plates 110, 210; first chambers 111, 211; liquid inlet channel 112; liquid outlet channel 113;

[0045] Second body 120, 220; Second chamber 121, 221; Gas passage 122;

[0046] Culture membranes 130 and 230; substrate 131; strain electrode 132; deformable part 1321; non-deformable part 1322; signal line 133; protective layer 134;

[0047] Third body 240; Third chamber 241;

[0048] Actuator membrane 250. Detailed Implementation

[0049] The embodiments of this application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0050] In the description of this application, it should be understood that the orientation descriptions, such as up, down, front, back, left, right, etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0051] In the description of this application, "several" means one or more, "more than" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0052] In the description of this application, unless otherwise expressly defined, terms such as "setup," "installation," and "connection" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this application in conjunction with the specific content of the technical solution.

[0053] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0054] There is a need for strain monitoring in organ-on-a-chip technology, but the strain monitoring methods used in related technologies have certain shortcomings:

[0055] (1) Most of the current organ-on-a-chip sensing structures (such as electrodes) used to monitor strain do not have stretchability and cannot directly monitor strain.

[0056] (2) The manufacturing process of flexible circuit boards containing electrodes in related technologies is complex, which leads to cumbersome operation and increased costs.

[0057] (3) Flexible circuit boards are difficult to miniaturize and miniaturize, and are difficult to integrate into the microchannels of organ-on-a-chip.

[0058] (4) Related technologies often only allow monitoring of a single culture chamber and do not achieve multi-channel monitoring;

[0059] (5) The electrodes have poor mobility, and usually one type of electrode is only suitable for organ-on-a-chip with a specific structure.

[0060] For example, Olivier T. Guenat et al. constructed a microimpedance computed tomography system that can monitor mechanical changes occurring in the alveolar barrier using impedance coplanar electrodes. In this scheme, a flexible circuit board (bendable but lacking tensile strength) and an actuation membrane from an organ-on-a-chip are stacked together. Electrodes on the flexible circuit board can monitor the impedance of the solution, indirectly monitoring the strain of the culture membrane. A certain gap (1 mm) is maintained between the flexible circuit board and the culture membrane to avoid affecting the deflection deformation of the culture membrane (approximately 600 μm). Light-transmitting holes are also formed on the flexible circuit board, aligned with the cell growth area on the culture membrane to avoid interfering with cell observation.

[0061] However, this method can only indirectly obtain strain data of the culture membrane, which is prone to error, and it is only applicable to specific organ-on-a-chip, with poor transferability.

[0062] In response to this situation, this application provides an in-situ monitoring method to more accurately monitor the strain of samples.

[0063] The in-situ monitoring method provided in this application includes the following steps:

[0064] Step S110: The culture membrane of the organ-on-a-chip receives cells, wherein the culture membrane integrates a stretchable strain electrode;

[0065] Step S120: Culture cells in the culture chambers on both sides of the culture membrane;

[0066] Step S130: Control the pressure in the culture chamber to change, thereby agitating the culture membrane;

[0067] Step S140: Acquire the electrical signal output of the strain electrode to monitor the strain of the culture membrane.

[0068] The testing principle of the in-situ monitoring method is as follows: When pressure P (positive or negative pressure) is applied to the culture membrane through the medium (liquid or gaseous medium) in the culture chamber of the organ-on-a-chip, it causes the culture membrane to move. The surface area of ​​the strain electrode changes (i.e., strain), and the change in its output signal (electrical signal) can be monitored simultaneously. When a certain pressure is applied, the strain electrode will generate a corresponding strain, and due to its electromechanical characteristics, it will output an electrical signal. Therefore, the correspondence between the strain of the culture membrane and the electrical signal can be calibrated. By monitoring the change in the electrical signal, real-time in-situ monitoring of the culture membrane on the organ-on-a-chip can be achieved.

[0069] According to the in-situ monitoring method provided in this application, by using a culture membrane with strain electrodes, the strain electrodes can collect strain data of the culture membrane in real time and accurately, so that the organ-on-a-chip can monitor the strain of the culture membrane in-situ, non-destructively and accurately.

[0070] Furthermore, since the deflection of the culture membrane is directly reflected by the tensile deformation of the strain electrode, the in-situ monitoring method is more flexible and has a wider range of applications. For example, it does not require that the organ-on-a-chip must have an actuation membrane, and the shape of the culture chamber and the composition of the culture medium are not restricted. The organ-on-a-chip can be used to simulate liquid-liquid interfaces as well as gas-liquid interfaces (such as the air-blood barrier of the alveoli), and so on.

[0071] Furthermore, multiple strain electrodes can be integrated onto the culture membrane, each corresponding to a different culture chamber. This facilitates the integration and arraying of culture chambers within organ-on-a-chip systems, enabling large-scale cell culture and experiments. The size and performance of the strain electrodes can be customized, allowing for compatibility with organ-on-a-chip systems of various structures and sizes.

[0072] Understandably, electrical signals need to be converted to obtain strain data. Although theoretically the conversion relationship between electrical signals and strain can be calculated based on the relevant parameters of the culture membrane (size, material, etc.), it is quite complicated to directly derive the conversion function between electrical signals and strain data due to manufacturing errors and the uneven distribution of strain electrodes on the culture membrane (they do not occupy the entire culture membrane).

[0073] Therefore, the in-situ monitoring method may optionally include calibrating the stress-strain relationship of the culture membrane before receiving cells. Calibration allows for the acquisition of a more accurate conversion table, thereby enabling better strain monitoring.

[0074] On the other hand, in addition to strain monitoring, other relevant information needs to be monitored during cell culture, such as cell distribution and growth status on the culture membrane. In some experiments, light stimulation also needs to be applied to the cultured cells. Therefore, strain electrodes can optionally be light-transmitting. This allows in-situ monitoring methods to observe cell growth during cell culture, while also meeting the light irradiation requirements in some special scenarios, thus providing a guarantee for physiological and pathological research on organ-on-a-chip.

[0075] Transparency can be achieved in different ways. For example, transparent or translucent materials can be used to make strain electrodes, or perforated structures can be created on the strain electrodes to allow light to pass through.

[0076] To implement the in-situ monitoring method provided in this application, this application also provides an organ-on-a-chip. This application further discloses the application of the organ-on-a-chip in in-situ cell monitoring.

[0077] Reference Figure 1 and Figure 2 The organ-on-a-chip according to the first aspect of this application includes a first sheet 110, a second sheet 120 and a culture membrane 130. The first sheet 110 forms a first chamber 111, the second sheet 120 forms a second chamber 121, and the culture membrane 130 is located between the first sheet 110 and the second sheet 120, separating the first chamber 111 and the second chamber 121. The culture membrane 130 includes a substrate 131 and a strain electrode 132, the strain electrode 132 is disposed on the substrate 131, and the substrate 131 and the strain electrode 132 are capable of stretching and deformation.

[0078] The first chamber 111 and the second chamber 121 are the cell culture chambers. According to the organ-on-a-chip provided in this application, by using a strain electrode 132 that can be stretched and deformed, and integrating the strain electrode on the culture membrane 130, the strain electrode 132 can more realistically and accurately collect the strain data of the culture membrane 130, so that the organ-on-a-chip can more accurately monitor the strain of the sample.

[0079] Organ-on-a-chip can include multiple strain electrodes 132 simultaneously, thereby enabling multi-channel monitoring.

[0080] It is understood that in related technologies, the tensile deformation of the culture membrane 130 can be achieved through two-dimensional tensile deformation and three-dimensional expansion deformation. The culture membrane 130 of the integrated strain electrode 132 of this application is applicable to both methods and can be implemented even without the actuation membrane, exhibiting good portability. In the embodiments of the first aspect of this application, a culture membrane 130 capable of three-dimensional expansion deformation is used as an example for description.

[0081] Optionally, the strain electrode 132 can be further divided into a deformable portion 1321 and a non-deformable portion 1322, wherein the deformable portion 1321 refers to the portion that will undergo tensile deformation during sample culture, and the non-deformable portion 1322 refers to the portion that will not undergo tensile deformation during sample culture.

[0082] For example, refer to Figure 2 and Figure 3 The culture membrane 130 is sandwiched between the first sheet 110 and the second sheet 120. Only the suspended portion of the culture membrane 130 (i.e., the portion used for culturing the sample) can undergo three-dimensional expansion and contraction under pressure, while the other portions of the culture membrane 130, although capable of tensile deformation, do not actually undergo tensile deformation. Correspondingly, refer to... Figure 3 and Figure 4 The deformable part 1321 refers to the part of the strain electrode 132 that is suspended and not fixed (that is, the part located within the range of the first chamber 111 and aligned with the first chamber 111), while the rest of the strain electrode 132 is the non-deformable part 1322.

[0083] In some scenarios, the culture membrane 130 needs to realistically simulate the growth environment of cells and tissues in vivo. For example, during in vitro culture of alveoli, the culture membrane 130 is used to simulate the air-blood barrier between alveoli and pulmonary microvessels. By applying pressure to the culture membrane 130, it expands and contracts, simulating the normal human breathing process. In this case, the deformation of the culture membrane 130 needs to be as close as possible to the actual deformation of the alveoli.

[0084] Stiffness is a parameter used to measure the deformation capacity of a material. In the organ-on-a-chip of this application, since the culture membrane 130 has both strain electrode 132 and substrate 131, the stiffness of the two is difficult to match perfectly. The area with strain electrode 132 and the area without strain electrode 132 often have inconsistent stiffness, resulting in uneven deformation and a poor simulation effect of alveoli.

[0085] Therefore, the stiffness of the deformed portion 1321 and the stiffness of the substrate 131 can be further limited to within ±5%, thereby controlling the stiffness deviation of different regions within an acceptable range and reducing the degree of unevenness in the deformation of the culture membrane 130.

[0086] Specifically, the stiffness of the deformation part 1321 can be adaptively adjusted by designing the shape and size of the deformation part 1321 according to the specific deformation mode (two-dimensional deformation or three-dimensional deformation) of the culture membrane 130.

[0087] The non-deformable part 1322 can be made of the same material with elastic deformation capability as the deformable part 1321, or it can be made of a different material than the deformable part 1321, such as a material without elastic deformation capability. For design and manufacturing considerations, the non-deformable part 1322 and the deformable part 1321 are usually made of the same material.

[0088] Understandably, some related technologies require the illumination of samples. For example, it may be necessary to observe the growth of samples using an optical microscope, or to trigger fluorescence or other reactions in samples through light.

[0089] Optionally, the culture membrane 130 is light-transmitting, thus facilitating light exposure and observation.

[0090] Specifically, on the one hand, the deformable part 1321 can have a hole structure, through which light can pass, thereby reducing the obstruction of light by the deformable part 1321.

[0091] It should be noted that the pore structure in this application can be a pore structure at the macroscopic scale or a pore structure at the microscopic scale.

[0092] For example, when metal nanowires are used as the material for strain electrodes, a porous structure is formed between the crisscrossing metal nanowires at the microscopic level. By changing the wire diameter and density of the metal nanowires, a strain electrode 132 with suitable stiffness and light transmittance can be obtained.

[0093] Alternatively, the deformable part 1321 can be designed in a grid shape on a macroscopic scale, in which case the grid serves as a perforated structure.

[0094] Understandably, the microscopic porous structure allows light to fully illuminate all parts of the culture membrane on the shaded side, which is more effective in situations where illumination is required during culture (such as photostimulation experiments, fluorescence analysis, etc.) and samples need to be cultured on both sides of the culture membrane. The macroscopic porous structure has a larger size, making it suitable for observing samples on the culture membrane 130 under a microscope.

[0095] In situations where necessary, combining macroscopic and microscopic porous structures can achieve better in vitro culture results.

[0096] For example, refer to Figure 5 , Figure 5A physical diagram of an organ-on-a-chip according to a first aspect embodiment of this application is shown. It can be observed that the strain electrode 132 includes electrode wires. On the one hand, the electrode wires themselves have tiny perforations, that is, relatively small microscopic pore structures. On the other hand, the electrode wires extend in a tortuous manner, with gaps between adjacent segments of the electrode wires, forming relatively large pore structures. Therefore, it is possible to clearly photograph cell samples growing on the culture membrane 130 through the macroscopic pore structure, and also to utilize the microscopic pore structure to allow cells located on the backlight side of the strain electrode 132 to receive sufficient light.

[0097] Obviously, in addition to the strain electrode 132 having a porous structure, the substrate 131 needs to be made of a transparent material so as not to obscure the porous structure.

[0098] To meet the requirements of tensile deformation, electrical signal transmission, and light transmittance, the material of the strain electrode 132 may optionally include one or more of graphene, carbon nanotubes, carbon fibers, and MXene.

[0099] Understandably, due to the ease of processing and high signal detection sensitivity of traditional metal materials, most signal detection systems choose to use metal materials as electrodes. However, metal materials have disadvantages in terms of biocompatibility. Therefore, this application preferably uses materials with better biocompatibility, such as laser-induced graphene (LIG), as the strain electrode 132. In addition, this application also adds a patterning step, thereby enhancing the sensitivity of the strain electrode 132.

[0100] To meet the requirements of tensile deformation, light transmittance, and the placement of strain electrodes 132, the substrate 131 may optionally be made of one or more of PDMS, dual-network hydrogel, and elastic gel.

[0101] Understandably, the electrical signal generated by the strain electrode 132 needs to be processed by an external terminal. Optionally, the culture membrane 130 also includes a signal line 133, which is connected to the strain electrode 132 and extends out of the substrate. The signal line 133 is used to connect to the external terminal. Since tensile deformation capability is not required, the signal line 133 can be a wire commonly used in related technologies, such as copper wire or silver wire.

[0102] Optionally, refer to Figure 4 The connection between signal line 133 and strain electrode 132 is covered with a protective layer 134. The protective layer 134 is used to protect the connection from corrosion, thereby ensuring the stability of signal transmission. Optionally, the protective layer 134 can be made of materials commonly used in related technologies such as PDMS, Ecoflex, and Dragon Skin.

[0103] As mentioned above, depending on the different in vitro simulation requirements, organ-on-a-chip can use different methods to culture samples. For example, samples can be cultured on one side of the culture membrane 130 or on both sides of the culture membrane 130. In addition, organ-on-a-chip can also use different methods to actuate the culture membrane 130.

[0104] For example, in an embodiment of the first aspect, the first chamber 111 can be used to culture cells, and the second chamber 121 can be used to actuate the culture membrane 130.

[0105] Specifically, in order to culture cells, the first sheet 110 may include an inlet channel 112 and an outlet channel 113. The inlet channel 112 is connected to the first chamber 111 and is used to input culture medium. The outlet channel 113 is connected to the first chamber 111 and is used to collect waste liquid.

[0106] After cells are seeded on the side of the culture membrane 130 facing the first chamber 111, culture medium is continuously supplied to the cells through the inlet channel 112 and the outlet channel 113, thereby providing a suitable growth environment for the cells.

[0107] The inlet channel 112 and outlet channel 113 need to extend through the organ-on-a-chip to facilitate connection to external devices. Figure 2 In this embodiment, the liquid inlet channel 112 and the liquid outlet channel 113 extend from the first sheet 110. The liquid inlet channel 112 and the liquid outlet channel 113 only pass through the first sheet 110. In some other examples, the liquid inlet channel 112 and the liquid outlet channel 113 may also extend from other locations (e.g., the second sheet 120). This application does not limit this.

[0108] In order to actuate the culture membrane 130, the second sheet 120 may include a gas channel 122, which is connected to the second chamber 121 and is used to regulate the gas pressure in the second chamber 121.

[0109] By changing the pressure in the second chamber 121, a force can be applied evenly to the surface of the culture membrane 130, thereby causing the culture membrane 130 to deform more uniformly.

[0110] To avoid the strain electrode 132 stimulating normal cell growth, it can be positioned on the side of the culture membrane 130 near the second chamber 121. Alternatively, the surface of the culture membrane 130 can be modified with biomolecules.

[0111] For example, refer to Figure 6 This application also provides a second aspect of the embodiment, the difference between the organ-on-a-chip in the second aspect and the organ-on-a-chip in the first aspect is that the culture membrane 230 is used to culture cells on both sides, and the first chamber 211 and the second chamber 221 need to be circulated with culture medium.

[0112] Optionally, the organ-on-a-chip also includes a third sheet 240 and an actuation membrane 250, the third sheet 240 forming a third chamber 241, the actuation membrane 250 being located between the second sheet 220 and the third sheet 240, and the actuation membrane 250 separating the second chamber 221 and the third chamber 241.

[0113] Gas can be introduced into the third chamber 241. First, the pressure change in the third chamber 241 causes the actuation membrane 250 to deform. Then, the deformation of the actuation membrane 250 causes the pressure change in the second chamber 221, thereby transmitting the pressure to the culture membrane 130, causing the culture membrane 130 to deform accordingly.

[0114] In addition to the two culture and actuation methods shown above, organ-on-a-chip can also employ other design methods, which will not be elaborated here. Furthermore, technical features not mentioned in the embodiments of the second aspect, such as the specific design of the first sheet 210, the second sheet 220, and the culture membrane 230, can be referred to the embodiments of the first aspect, and will not be elaborated here.

[0115] It goes without saying that the organ-on-a-chip of the second aspect embodiment can also be used to implement the in-situ monitoring method of this application.

[0116] It should be noted that one of the key aspects of the in-situ monitoring method and organ-on-a-chip of this application is how to fabricate a culture membrane 130 that meets the requirements. Therefore, this application also provides a method for fabricating the culture membrane 130, which helps to more easily fabricate a stretchable strain electrode 132 and a culture membrane 130 that meet the requirements.

[0117] Exemplarily, the method for manufacturing the culture membrane 130 includes the following steps:

[0118] Step S210: Form strain electrode 132 on the carrier.

[0119] For example, the strain electrode 132 may be laser-induced graphene (LIG). In this example, forming the strain electrode 132 on the carrier may include forming the laser-induced graphene on the PI film by laser reduction.

[0120] Step S220: Form a substrate 131 on the carrier that is bonded to the strain electrode 132.

[0121] The material of substrate 131 needs to be stretchable, transparent, and biocompatible to meet the requirements of cell growth and observation, and to be able to be stretched over a large range without damage. For example, substrate 131 can be PDMS, and substrate 131 and strain electrode 132 can be combined by infiltration.

[0122] Specifically, forming a substrate 131 on the carrier and bonding it with the strain electrode 132 may include spin-coating a PDMS solution onto the carrier and curing the PDMS solution.

[0123] Using a permeation method, PDMS penetrates into the gaps of laser-induced graphene during spin coating, thus bonding them tightly together.

[0124] Of course, in addition to forming the strain electrode 132 through a carrier, the substrate 131 and the strain electrode 132 can also be combined by methods such as evaporation, sputtering, penetration transfer, and transfer printing. For example, the strain electrode 132 can be obtained by sputtering or evaporation on the surface of the PDMS material substrate 131, or by spin coating on the substrate 131. The substrate 131 can also be a flexible and stretchable material such as a dual-network hydrogel or an elastic gel. This application does not limit this.

[0125] It should be noted that in methods such as sputtering and transfer, the bonding between the strain electrode 132 and the substrate 131 is not tight enough. However, by using the permeation method, the bonding between the strain electrode 132 and the substrate 131 can be effectively enhanced, ensuring the accuracy of monitoring subtle stress changes in the cell layer. At the same time, using the permeation method, the thickness of the culture membrane 130 carrying the strain electrode 132 can be controlled at 100 μm, improving the sensitivity of the electrode and meeting the needs of more scenarios.

[0126] Step S230: Remove the carrier.

[0127] After the PDMS solution is cured, the carrier is peeled off to obtain a culture film 130 in which the substrate 131 and the strain electrode 132 are bonded together.

[0128] Optionally, the method for fabricating the culture membrane 130 further includes step S240: forming a signal line 133 connected to the strain electrode 132.

[0129] For example, the signal line 133 can be made of copper wire, and the signal line 133 and the strain electrode 132 can be connected by conductive silver paste. The signal line 133 is led out of the substrate 131 to facilitate the connection of the monitoring line.

[0130] Furthermore, the method for fabricating the culture membrane 130 also includes step S250: forming a protective layer 134 covering the connection portion of the signal line 133 and the strain electrode 132.

[0131] In summary, in this application:

[0132] 1. By transferring laser-induced graphene to the surface of a flexible and stretchable elastic material (substrate 131), a flexible and stretchable strain electrode 132 is fabricated, which helps to realize the fabrication of a fully flexible organ-on-a-chip. Moreover, the fabrication method is simple. In the laser induction process, graphene can be selectively induced on the carrier surface by adjusting the irradiation area of ​​the laser, thereby flexibly forming strain electrodes 132 of different shapes, which facilitates the patterning of strain electrodes 132.

[0133] 2. By designing the size of laser-induced graphene, it is possible to miniaturize and micronize the strain electrode 132, thereby integrating it into the culture membrane 130 of the organ-on-a-chip. Furthermore, the arrayed strain electrode 132 enables multi-channel monitoring of strain in the organ-on-a-chip.

[0134] 3. The stretchable strain electrode 132 of this application is used to monitor strain in organ-on-a-chip. It has a sensitive response to different strains and can read out the strain signal in real time, realizing in-situ real-time monitoring of strain.

[0135] 4. The organ-on-a-chip based on the strain electrode 132 of laser-induced graphene-elastic material, as a tool for in-situ strain monitoring, has potential application prospects in monitoring weak strains due to its simple fabrication process and ease of miniaturization, integration, and arraying.

[0136] Understandably, after fabricating the culture membrane 130 using the same method, it needs to be assembled with structures such as the first sheet 110 and the second sheet 120 (e.g., through bonding) to form an organ-on-a-chip. Next, an in-situ monitoring system can be built based on the organ-on-a-chip to achieve real-time monitoring.

[0137] Taking the organ-on-a-chip of the first aspect embodiment as an example, specifically, the gas channel 122 is connected to the trachea and the pressure input is controlled by a pressure controller (e.g., a control system integrating an air compressor and a vacuum pump); the strain electrode 132 is connected to an external signal acquisition device (digital source meter / digital multimeter) through the signal line 133 to collect electrical signal output under different pressures in real time; the liquid inlet channel 112 injects cells and their required nutrients, and the liquid outlet channel 113 discharges waste liquid.

[0138] While controlling the input pressure with a pressure controller, a digital multimeter is used to simultaneously test and collect the real-time electrical signal of the strain electrode 132. The electrical signal can be output to the terminal (computer) through the equipment's software, and then the real-time strain value can be calculated, thereby realizing in-situ real-time monitoring of the strain of the culture membrane 130 on the organ-on-a-chip and the sample on it.

[0139] Figure 7Part (a) shows the relationship between the vertical displacement and strain of the culture membrane 130 in the organ-on-a-chip. The inset shows the principle and method of strain calculation, which is to characterize the strain by the rate of change of the surface area of ​​the culture membrane 130.

[0140] When a negative pressure is applied to the culture membrane 130 through the second chamber 121, the surface of the culture membrane 130 bends downward and deforms. Figure 7 Part (b) illustrates the relationship between applied pressure and vertical displacement of the culture membrane 130 surface. From this, a relationship between applied pressure and strain can be constructed, such as... Figure 7 As shown in section (c).

[0141] Meanwhile, due to the electromechanical properties of the strain electrode 132, surface deformation of the culture membrane 130 will cause a change in the resistance of the strain electrode 132. Figure 8 Part (a) shows the relationship between the applied stress and the rate of change of resistance of strain electrode 132. Figure 8 Part (b) further details the recorded changes in resistance and pressure during the input pressure process, showing that these changes are synchronous. This allows for the establishment of a correspondence between strain and electrical signal. Therefore, monitoring changes in the electrical signal can instantly, non-destructively, and non-invasively reflect changes in the strain of the culture membrane 130, thereby achieving in-situ real-time monitoring of the strain of the culture membrane 130.

[0142] Figure 9 The strain change curve in a monitoring experiment is shown. According to the experimental results, the organ-on-a-chip is suitable for monitoring strain of less than 1%. The research results in the literature show that the volume of myocardial tissue changes by 2-4% during contraction. Therefore, the experimental results verify that the organ-on-a-chip of this application can realize in situ monitoring of cellular strain.

[0143] In some alternative embodiments, the functions / operations mentioned in the block diagrams may not occur in the order shown in the operation diagrams. For example, depending on the functions / operations involved, two consecutively shown blocks may actually be executed substantially simultaneously, or the blocks may sometimes be executed in reverse order. Furthermore, the embodiments presented and described in the flowcharts of this application are provided by way of example to provide a more comprehensive understanding of the technology. The disclosed methods are not limited to the operations and logic flows presented herein. Alternative embodiments are contemplated in which the order of various operations is changed and sub-operations described as part of a larger operation are executed independently.

[0144] Although embodiments of this application have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the claims and their equivalents.

Claims

1. An in-situ monitoring method for organ-on-a-chip, characterized in that, include: The organ-on-a-chip has a culture membrane that receives cells, wherein the culture membrane is integrated with a stretchable strain electrode. The cells are cultured in the culture chambers on both sides of the culture membrane; The pressure in the culture chamber is controlled to change, thereby agitating the culture membrane; The electrical signal output of the strain electrode is collected to monitor the strain of the culture membrane.

2. The in-situ monitoring method according to claim 1, characterized in that, The in-situ monitoring method further includes calibrating the stress-strain relationship of the culture membrane before receiving the cells.

3. The in-situ monitoring method according to claim 1, characterized in that, The strain electrode is transparent.

4. An organ-on-a-chip, characterized in that, include: The first piece forms the first chamber; The second piece forms the second chamber; A culture membrane is located between a first sheet and a second sheet, the culture membrane separating the first chamber and the second chamber, the culture membrane including a substrate and a strain electrode disposed on the substrate, the substrate and the strain electrode being capable of tensile deformation.

5. The organ-on-a-chip according to claim 4, characterized in that, The strain electrode includes a deformation portion located within the first chamber.

6. The organ-on-a-chip according to claim 5, characterized in that, The deviation between the stiffness of the deformed part and the stiffness of the substrate shall not exceed ±5%.

7. The organ-on-a-chip according to claim 4, characterized in that, The culture membrane is transparent.

8. The organ-on-a-chip according to claim 7, characterized in that, The strain electrode has a porous structure.

9. The organ-on-a-chip according to claim 7, characterized in that, The substrate is made of a transparent material.

10. The organ-on-a-chip according to claim 4, characterized in that, The culture membrane also includes a signal line connected to the strain electrode, the signal line extending out of the substrate, and the signal line being used to connect to an external terminal.

11. The organ-on-a-chip according to claim 10, characterized in that, The portion of the signal line that connects to the strain electrode is covered with a protective layer.

12. The organ-on-a-chip according to claim 4, characterized in that, The strain electrode is made of one or more of the following materials: graphene, carbon nanotubes, carbon fibers, and MXene.

13. The organ-on-a-chip according to claim 4, characterized in that, The substrate material includes one or more of PDMS, dual-network hydrogel, and elastic gel.

14. The organ-on-a-chip according to claim 4, characterized in that, The first chamber is used to culture cells, and the second chamber is used to actuate the culture membrane.

15. The organ-on-a-chip according to claim 14, characterized in that, The organ-on-a-chip includes an inlet channel and an outlet channel. The inlet channel is connected to the first chamber and is used to input culture medium. The outlet channel is connected to the first chamber and is used to collect waste liquid.

16. The organ-on-a-chip according to claim 14, characterized in that, The organ-on-a-chip includes a gas channel that communicates with the second chamber and is used to regulate the gas pressure in the second chamber.

17. The organ-on-a-chip according to claim 14, characterized in that, The strain electrode is located on the side of the culture membrane closer to the second chamber.

18. The organ-on-a-chip according to claim 4, characterized in that, The organ-on-a-chip also includes a third sheet and an actuation membrane, the third sheet forming a third chamber, the actuation membrane being located between the second sheet and the third sheet, and the actuation membrane separating the second chamber and the third chamber.

19. A method for preparing a culture membrane, characterized in that, include: Strain electrodes are formed on the carrier; A substrate is formed on the carrier and bonded to the strain electrode; Remove the carrier.

20. The method for preparing a culture membrane according to claim 19, characterized in that, The method for fabricating the culture membrane further includes forming a signal line connected to the strain electrode.

21. The method for preparing a culture membrane according to claim 20, characterized in that, The method for fabricating the culture membrane further includes forming a protective layer covering the connection between the signal line and the strain electrode.

22. The method for preparing a culture membrane according to claim 19, characterized in that, Forming the strain electrode on the carrier includes forming laser-induced graphene on a PI film by laser reduction.

23. The method for preparing a culture membrane according to claim 22, characterized in that, Forming a substrate on the carrier and bonding it to the strain electrode includes spin-coating a PDMS solution on the carrier; The PDMS solution is solidified.

24. The application of the organ-on-a-chip according to any one of claims 4 to 18 in in situ monitoring.

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