Multi-organ chip as well as preparation method and application method thereof

Abstract

The invention provides a multi-organ chip as well as a preparation method and an application method thereof. The multi-organ chip comprises a chip body and at least two cell culture inserts, the chip body comprises a lower clamping plate, a substrate layer, a micro-channel layer, a cavity layer and an upper clamping plate assembly which are stacked from bottom to top, the cavity layer is provided with at least two cavities at intervals, the micro-channel layer is provided with micro-channel holes which are in one-to-one correspondence with the cavities and are communicated with the cavities, and the upper clamping plate assembly is connected with the substrate layer. The substrate layer covers the bottom of the micro-channel layer, the chamber layer, the micro-channel layer and the substrate layer jointly form at least two chambers, and each chamber is used for accommodating a detachable cell culture insert; the upper clamping plate assembly is provided with a perfusion connector, the cavity layer is provided with perfusion holes, the micro-channel layer is provided with perfusion slits, the perfusion slits are communicated with the micro-channel holes through the perfusion holes, and the micro-channel layer is further provided with micro-channel slits communicated with every two adjacent cavities. On the premise that a radiation shielding device is not used, construction of partial cell radiation models is achieved, and plug-and-play of the cell culture plug-in is achieved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a multi-organ chip, its preparation method, and its application method. Background Technology

[0002] With the development of manned spaceflight technology, astronauts' stays in space have gradually changed from short-term flights to long-term flights. The radiation dose received by astronauts in space is constantly increasing, and the threat of space ionizing radiation to their health has become a critical issue that must be addressed. Radiation not only damages irradiated cells and tissues but also causes non-target effects in unirradiated cells and tissues. These effects can generally be divided into radiation-induced bystander effects (RIBE) and radiation-induced abscopal effects (RIAE).

[0003] Research on real-world space radiation environments requires the use of experimental satellites and space stations, which is costly and limited by the scarcity of such opportunities, making it difficult to meet research needs. Furthermore, research on non-target effects of radiation requires the simultaneous construction of both irradiated and unirradiated cell models, and the use of conditioned medium transfer to facilitate their interaction. However, in a real-world space environment, it is difficult to achieve radiation shielding for some cells and construct unirradiated cell models. Therefore, research on non-target effects induced by space ionizing radiation is generally conducted in ground-based laboratories.

[0004] In ground-based laboratories, researchers often use in vitro models to study RIAEs or RIBEs, such as the Transwell assay and conditioned medium transfer assay, simulating the space radiation environment using ground-based radiation sources. However, the conditioned medium transfer assay can only study cumulative non-target effects over a period of time, which differs significantly from the actual real-time interactions between cells or tissues in vivo. The Transwell assay can construct a static model of real-time cell interactions, which is inconsistent with the real-world situation in vivo. Furthermore, in vivo models of complex spaces can be constructed using animals such as rats and mice, and are also commonly used in RIAE and RIBE studies. However, in vivo models involve complex interactions between multiple organs, making it difficult to accurately evaluate specific interactions. Therefore, existing traditional in vitro / in vivo models commonly used in studies of non-target effects of space radiation all have limitations.

[0005] Organ-on-a-chip technology is a type of perfusion-enabled, highly biomimetic three-dimensional cell culture device that can construct biomimetic organs in vitro that closely resemble those in vivo. Following the successful construction of the first lung organ-on-a-chip by Donald et al. in 2010, various other organ-on-a-chip technologies have been successfully developed, including blood-brain barrier chips and kidney chips. Currently, organ-on-a-chip technology has become an important tool in life science research. Furthermore, the various organs in the human body do not exist in isolation; there are extensive and close interactions between them. Multi-organ-on-a-chip technology integrates multiple organs based on organ-on-a-chip technology to construct multiple interacting human organs, overcoming the limitations of traditional in vitro models that differ significantly from the physiological structure of tissues and can only measure cumulative effects.

[0006] Multi-organ-on-a-chip technology can overcome the above-mentioned shortcomings, making up for the large differences between traditional in vitro models and tissue physiological structures and the inability to measure only cumulative effects. It has great potential to construct high-level research models of ionizing radiation-induced non-target effects. However, common multi-organ-on-a-chip technologies often require the construction of multiple organs or tissues and their fixation on a single chip before the experiment. Therefore, it is necessary to use radiation shielding devices or adjust the irradiation window of the particle accelerator to ensure that the radiation only irradiates the parts that need to be irradiated. These methods are often quite cumbersome. Summary of the Invention

[0007] To solve the above-mentioned technical problems, or at least partially solve them, the present invention provides a multi-organ chip and its preparation and application methods, which not only realizes the construction of a partial cell radiation model without the use of a radiation shielding device, but also enables the plug-and-play use of cell culture plug-ins.

[0008] The first aspect of the present invention provides a multi-organ chip, comprising a chip body and at least two cell culture plugs;

[0009] The chamber layer is provided with at least two cavities at intervals, the microchannel layer is provided with microchannel holes that correspond one-to-one with and communicate with the cavities, the base layer covers the bottom of the microchannel layer, the chamber layer, the microchannel layer and the base layer together constitute at least two chambers, each chamber can accommodate one of the cell culture plugs, the cell culture plugs are detachably inserted into the chambers, and the upper clamping plate assembly is provided with communication holes that correspond one-to-one with and communicate with the chambers;

[0010] The upper clamping plate assembly is provided with an irrigation connector, the chamber layer is provided with an irrigation hole corresponding to and communicating with the irrigation connector, the microchannel layer is provided with an irrigation slit corresponding to and communicating with the irrigation hole, the irrigation slit is communicating with the microchannel hole, and the microchannel layer is also provided with a microchannel slit communicating with two adjacent chambers.

[0011] Optionally, the lower clamping plate, the base layer, the microchannel layer, the chamber layer, and the upper clamping plate assembly are each provided with multiple mating holes, so as to fix them by inserting fastening bolts through the multiple mating holes and tightening fastening nuts on the fastening bolts.

[0012] Optionally, a flexible membrane is provided between the lower bottom surface of the upper clamping plate assembly and the upper surface of the chamber layer. The flexible membrane has a connecting hole that corresponds to and communicates with the irrigation hole, so that the culture medium injected from the irrigation connector enters the irrigation hole through the connecting hole.

[0013] Optionally, the upper clamping plate assembly includes a first upper clamping plate and a second upper clamping plate, with the first upper clamping plate stacked on top of the second upper clamping plate. The first and second upper clamping plates are respectively provided with two perfusion inlets and outlets, and each perfusion inlet and outlet is provided with a perfusion connector. The perfusion connector is used to connect to an external conduit to realize the perfusion of the culture medium through the perfusion connector.

[0014] Optionally, the cell culture plug includes a carrier and a cell culture scaffold;

[0015] The cell culture scaffold is used to culture adherent cells and construct an in vitro model of a physiological system. The carrier has a receiving cavity with an opening at the top, and the cell culture scaffold is placed in the receiving cavity.

[0016] The upper opening of the carrier has a support edge extending outward in the circumferential direction, and the cell culture plug is supported on the upper surface of the chamber layer by the support edge.

[0017] Optionally, the carrier includes a first annular sheet, a porous filter membrane, a second annular sheet, a third annular sheet, and a fourth annular sheet stacked from bottom to top;

[0018] The porous filter membrane is fixed between the first annular plate and the second annular plate. After the cell culture plug is inserted into the chamber, the side of the porous filter membrane corresponds to the microchannel slit, which is used to separate the cells in the cell culture plug from the fluid in the chamber and to construct the endothelial barrier.

[0019] The second, third, and fourth annular plates together form the receiving cavity. The diameter of the cell culture scaffold is smaller than the inner diameter of the third and fourth annular plates but larger than the inner diameter of the second annular plate, so that the cell culture scaffold can be suspended above the porous filter membrane.

[0020] The outer diameters of the third, second, and first annular plates are smaller than the inner diameter of the cavity in the chamber layer, and the outer diameter of the fourth annular plate is larger than the inner diameter of the cavity in the chamber layer. The fourth annular plate forms the supporting edge, and the cell culture insert is supported on the upper surface of the chamber layer by the fourth annular plate.

[0021] A second aspect of the present invention provides a method for fabricating a multi-organ chip, for fabricating a multi-organ chip as described in any of the preceding claims, comprising the following steps:

[0022] Fabrication of the chip substrate;

[0023] Preparation of cell culture modules;

[0024] The chip preparation body includes:

[0025] Prepare an upper clamping plate assembly and a lower clamping plate, and provide an infusion connector on the upper clamping plate assembly;

[0026] Preparation of the chamber layer;

[0027] Preparation of microchannel layer and substrate layer;

[0028] The microchannel layer, the basal layer, and the chamber layer are bonded together so that the chamber layer, the microchannel layer, and the basal layer together form at least two chambers.

[0029] Optionally, the preparation of the upper clamping plate assembly and the lower clamping plate, and the provision of an irrigation connector on the upper clamping plate assembly, includes:

[0030] Machining the first upper clamping plate, the second upper clamping plate, and the lower clamping plate;

[0031] The first upper clamping plate and the second upper clamping plate were bonded together using epoxy resin potting compound.

[0032] Use double-sided tape to fix the irrigation connector at the irrigation inlet and outlet of the first upper clamp and the second upper clamp, and use epoxy resin potting compound to seal around the irrigation connector.

[0033] Using rigid clamps as an aid, pressure is applied by setting fastening bolts and fastening nuts on the first upper clamping plate and the second upper clamping plate to make the adhesion firm.

[0034] Optionally, the preparation of the chamber layer includes:

[0035] Polydimethylsiloxane was selected as the material for the chamber layer. The curing agent and the main body were mixed and stirred according to the recommended mixing ratio of polydimethylsiloxane at a preset mass ratio to obtain a polydimethylsiloxane premix.

[0036] Pour a premix of polydimethylsiloxane of a predetermined thickness into a polystyrene mold;

[0037] After curing, the polydimethylsiloxane block is obtained by demolding.

[0038] A predetermined number of cavities are cut into the polydimethylsiloxane block using a die cutter of the first preset diameter to obtain a cavity layer;

[0039] The top of the chamber layer is sealed using a polypropylene adhesive sealing film.

[0040] Optionally, the fabrication of the microchannel layer and the substrate layer includes:

[0041] Using laser engraving technology, a channel positive mold pattern is cut out from a polymethyl methacrylate sheet to obtain a polymethyl methacrylate channel positive mold.

[0042] A polystyrene mold was selected as the substrate, epoxy resin potting compound was dripped in, and the polymethyl methacrylate channel positive mold was placed on top and pasted.

[0043] After curing, a microchannel layer mold and a substrate layer mold are obtained;

[0044] Polydimethylsiloxane is selected as the material for the microchannel layer and the base layer. The polydimethylsiloxane premix, which is prepared by the curing agent and the main body in a preset mass ratio, is poured into the microchannel layer mold and the base layer mold, so that the polydimethylsiloxane is at least higher than the top of the polymethyl methacrylate channel positive mold by a preset distance.

[0045] After curing, the microchannel layer and the base layer are obtained by demolding.

[0046] Optionally, the preparation of the cell culture plug includes:

[0047] Three polydimethylsiloxane discs with a first preset thickness and one with a second preset thickness were prepared using a petri dish;

[0048] Using die-cutting blades of a second and a third preset diameter on two polydimethylsiloxane discs of a first preset thickness, rings are cut out;

[0049] A porous terephthalic acid filter membrane with a diameter of a first preset length and a pore size of a second preset length is attached to the center of a polydimethylsiloxane disc of a first preset thickness. The two polydimethylsiloxane discs are then bonded together by plasma bonding, thereby fixing the porous filter membrane between the two polydimethylsiloxane discs of the first preset thickness. The porous filter membrane is made of at least one of polyethylene terephthalate, polycarbonate, polyester, polyimide, polyvinylidene fluoride, and polydimethylsiloxane.

[0050] A ring is cut out using a die-cutting blade with a second preset diameter and a fourth preset diameter on a polydimethylsiloxane disc of a second preset thickness. A disc is cut out using a die-cutting blade with a fifth preset diameter on another polydimethylsiloxane disc of a first preset thickness. The discs are then plasma bonded sequentially. The second preset diameter is greater than the fourth preset diameter, the fourth preset diameter is greater than the third preset diameter, and the fifth preset diameter is greater than the second preset diameter.

[0051] Use a scriber, die cutter, or punch to cut a hole in the center of the top polydimethylsiloxane disc to serve as an entry point for the cell culture scaffold, thus completing the processing of the cell culture insert.

[0052] A third aspect of the present invention provides a method for applying a multi-organ-on-a-chip, employing a multi-organ-on-a-chip as described in any of the preceding claims, comprising the following steps:

[0053] Cells are cultured in N cell culture modules. 1 to N-1 cell culture modules are irradiated to culture the irradiated cells, while the remaining cell culture modules are not irradiated to culture the unirradiated cells. N≥2.

[0054] All cell culture modules are assembled into the corresponding chambers of the multi-organ chip. Among them, the cell culture module with irradiated cells is used as the upstream cell culture module in the perfusion process, and the cell culture module with unirradiated cells is used as the downstream cell culture module in the perfusion process.

[0055] Culture medium is infused into the multi-organ chip via a perfusion connector, with a preset perfusion duration to simulate fluid flow and cell interactions in the in vivo environment.

[0056] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0057] The present invention provides a multi-organ chip, its preparation method, and its application method. The multi-organ chip includes a chip body and at least two cell culture inserts. The chip body includes a lower clamping plate, a base layer, a microchannel layer, a chamber layer, and an upper clamping plate assembly stacked from bottom to top. The chamber layer, the microchannel layer, and the base layer together form at least two chambers, each of which can accommodate a detachable cell culture insert. Thus, by seeding different types of cells onto different cell culture inserts, multiple organs or tissues can be simulated. The cell culture inserts are detachably inserted into the chambers of the chip body, rather than being directly fixed to the chip body, allowing staff to easily control and adjust the configuration of the multi-organ chip and achieve plug-and-play functionality for the cell culture inserts. At the same time, it avoids the cumbersome operation of using radiation shielding devices or adjusting the irradiation window of a particle accelerator when constructing a non-target effect research model of partial organ irradiation. Attached Figure Description

[0058] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0059] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0060] Figure 1 This is a schematic diagram of the exploded structure of a multi-organ chip provided in an embodiment of the present invention;

[0061] Figure 2 A schematic diagram of the assembly structure of a multi-organ chip provided in an embodiment of the present invention;

[0062] Figure 3 This is an exploded structural diagram of the cell culture plug-in for a multi-organ chip provided in an embodiment of the present invention.

[0063] Figure 4 This is a schematic diagram of the assembly structure of the cell culture plug-in for a multi-organ chip provided in an embodiment of the present invention;

[0064] Figure 5 This is a schematic flowchart of the multi-organ-on-a-chip fabrication method provided in an embodiment of the present invention;

[0065] Figure 6 This is a schematic diagram illustrating the process of preparing the upper clamping plate assembly, the lower clamping plate, and setting the infusion connector on the upper clamping plate assembly, as provided in an embodiment of the present invention.

[0066] Figure 7 This is a schematic diagram illustrating the preparation of the first upper clamping plate, the second upper clamping plate, and the lower clamping plate using the preparation method provided in the embodiments of the present invention;

[0067] Figure 8 This is a schematic diagram of the process for preparing the chamber layer provided in an embodiment of the present invention;

[0068] Figure 9 This is a schematic diagram of the process for preparing the microchannel layer and substrate layer provided in an embodiment of the present invention;

[0069] Figure 10 This is a schematic diagram of the process for preparing the cell culture plug-in according to an embodiment of the present invention;

[0070] Figure 11 This is a schematic diagram illustrating the manufacturing process of the cell culture plug provided in an embodiment of the present invention;

[0071] Figure 12This is a flowchart illustrating the multi-organ-on-a-chip application method provided in an embodiment of the present invention;

[0072] Figure 13 A schematic diagram showing the TEER value measurement results of BBB 48 hours after perfusion, provided in an embodiment of the present invention;

[0073] Figure 14 This is a schematic diagram illustrating the analysis results of THP-1 cell adhesion and cell viability provided in an embodiment of the present invention;

[0074] Figure 15 This is a schematic diagram illustrating the analysis results of relative RNA expression levels in THP-1 cells provided in an embodiment of the present invention.

[0075] Among them, 1. Lower clamping plate; 11. Docking hole; 2. Base layer; 3. Microchannel layer; 31. Microchannel hole; 32. Irrigation slit; 33. Microchannel slit; 4. Chamber layer; 41. Cavity; 42. Irrigation hole; 5. Second upper clamping plate; 51. Irrigation connector; 6. First upper clamping plate; 61. Connecting hole; 62. Irrigation inlet and outlet; 7. Cell culture insert; 71. First annular plate; 72. Porous filter membrane; 73. Second annular plate; 74. Third annular plate; 75. Cell culture scaffold; 76. Fourth annular plate. Detailed Implementation

[0076] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0077] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0078] The multi-organ chip, its preparation method, and its application method provided by the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0079] Reference Figure 1 The diagram shown is an exploded view of the multi-organ chip provided in an embodiment of the present invention; see reference. Figure 2 The diagram shown is a schematic diagram of the assembly structure of a multi-organ chip provided in an embodiment of the present invention.

[0080] Reference Figure 1 and Figure 2 As shown, some embodiments of the present invention provide a multi-organ chip, including a chip body and at least two cell culture inserts 7. The chip body includes, from bottom to top, a lower clamping plate 1, a base layer 2, a microchannel layer 3, a chamber layer 4, and an upper clamping plate assembly.

[0081] The chamber layer 4 is provided with at least two cavities 41 spaced apart, the microchannel layer 3 is provided with microchannel holes 31 that correspond one-to-one with and communicate with the cavities 41, and the base layer 2 covers the bottom of the microchannel layer 3. The chamber layer 4, the microchannel layer 3, and the base layer 2 together constitute at least two chambers (see reference). Figure 2 In the text, a and b represent two chambers respectively. Each chamber is used to accommodate a cell culture plug 7. The cell culture plug 7 is detachably inserted into the chamber. The upper clamping plate assembly is provided with a communication hole 61 that corresponds to and communicates with the chamber.

[0082] By seeding different types of cells onto different cell culture plugs 7, various organs or tissues can be simulated. The cell culture plugs 7 are detachably inserted into the chambers of the chip body, rather than being directly fixed to the chip body, allowing staff to easily control and adjust the configuration of multi-organ chips, achieving plug-and-play functionality for the cell culture plugs 7. Furthermore, after the cell culture plugs 7 are inserted into the chambers, the upper clamping assembly can be stacked on top of the chamber layer 4 to fix the cell culture plugs 7 to the chip body. The upper clamping assembly has connecting holes 61 that correspond one-to-one with the chambers to ensure the breathability of the cell culture plugs 7, thereby ensuring the reliability of subsequent experimental work.

[0083] The upper clamping plate assembly is equipped with an infusion connector 51. The chamber layer 4 is equipped with an infusion hole 42 corresponding to and communicating with the infusion connector 51. The microchannel layer 3 is equipped with an infusion slit 32 corresponding to and communicating with the infusion hole 42. The infusion slit 32 communicates with the microchannel hole 31. The microchannel layer 3 is also equipped with a microchannel slit 33 connecting two adjacent chambers. The infusion connector 51 communicates with the microchannel hole 31 through the infusion hole 42 and the infusion slit 32, so as to infuse fluids such as culture medium into the chambers of the chip body through the infusion connector 51, and to realize the transport of substances between two adjacent chambers through the microchannel slit 33.

[0084] The multi-organ chip provided in this invention utilizes the chamber layer 4, microchannel layer 3, and base layer 2 of the chip body to construct at least two chambers. Each chamber can accommodate a detachable cell culture plug-in 7. In this way, by seeding different types of cells on different cell culture plug-ins 7, multiple organs or tissues can be simulated. Furthermore, the cell culture plug-in 7 is detachably inserted into the chamber of the chip body, rather than being directly fixed to the chip body. This achieves the plug-and-play function of the cell culture plug-in 7, allowing staff to easily control and adjust the configuration of the multi-organ chip. At the same time, it avoids the cumbersome operation of using radiation shielding devices or adjusting the particle accelerator irradiation window when constructing a non-target effect research model of partial organ irradiation.

[0085] Furthermore, after the cell culture plug-in 7 is inserted into the chamber of the chip body, the upper clamping plate assembly can be stacked on top of the chamber layer 4, and the lower clamping plate 1, the base layer 2, the microchannel layer 3, the chamber layer 4 and the upper clamping plate assembly can be connected and fixed with fastening bolts and fastening nuts, thereby achieving a stable assembly of the cell culture plug-in 7 on the chip body. The connecting hole 61 provided on the upper clamping plate assembly ensures the air permeability of the cell culture plug-in 7, thereby ensuring the reliability of subsequent experimental work.

[0086] It should be noted that, in specific implementation, refer to Figure 1 and Figure 2 As shown, the chip body can have two chambers, and correspondingly, the number of cell culture plug-ins 7 can be two, with each cell culture plug-in 7 being detachably inserted into one of the two chambers. Of course, the number of chambers on the chip body is not limited to two; it can also be three or more, and correspondingly, the number of cell culture plug-ins 7 can be three or more, with multiple cell culture plug-ins 7 being detachably inserted into multiple chambers.

[0087] It should be understood that when the number of chambers constructed on the chip body is three or more, the multiple chambers are not limited to being arranged in a straight line, that is, the multiple chambers are not limited to being located on the same straight line, but can also be arranged in a branching manner. For example, one chamber can be connected to the other two chambers through the microchannel slit 33. There is no specific limitation on the arrangement of the multiple chambers, as long as the material transport between different chambers can be realized through the microchannel slit 33.

[0088] In some embodiments, refer to Figure 1 and Figure 2 As shown, the lower clamping plate 1, the base layer 2, the microchannel layer 3, the chamber layer 4, and the upper clamping plate assembly are all provided with multiple docking holes 11, so as to fix them by inserting fastening bolts through the multiple docking holes 11 and tightening fastening nuts on the fastening bolts.

[0089] In practical implementation, the lower clamping plate 1, the base layer 2, the microchannel layer 3, the chamber layer 4, and the upper clamping plate assembly can have roughly the same outer contour (the outer contour of the upper clamping plate assembly can be slightly smaller). Multiple docking holes 11 can be spaced apart at the periphery of the lower clamping plate 1, the base layer 2, the microchannel layer 3, the chamber layer 4, and the upper clamping plate assembly. After the cell culture plug 7 is inserted into the chamber of the chip body, it can be clamped and fixed by inserting fastening bolts through the multiple docking holes 11 and tightening nuts on the fastening bolts. In this way, the fixing effect of the fastening bolts and nuts not only allows the chip body and the cell culture plug 7 to be assembled as one unit, but also allows for better fit and sealing between the stacked components, thereby ensuring the reliability of subsequent experimental work and avoiding leakage problems at the connection points of the chip body components and the connection points between the chip body and the cell culture plug 7.

[0090] Reference Figure 3 The diagram shown is an exploded view of the cell culture module of the multi-organ chip provided in an embodiment of the present invention; see reference. Figure 4 The diagram shown is a schematic diagram of the assembly structure of the cell culture plug-in for a multi-organ chip provided in an embodiment of the present invention.

[0091] In some embodiments, refer to Figure 4 As shown, the cell culture insert 7 includes a carrier and a cell culture scaffold 75. The carrier has a receiving cavity with an upper opening, and the cell culture insert 7 can be placed into the receiving cavity formed by the carrier through the upper opening. The cell culture scaffold 75 is used to culture adherent cells and construct an in vitro model of a physiological system. In addition to the cell culture scaffold 75, glass slides can also be used to culture adherent cells, and they have the same function as the cell culture scaffold 75 and can be substituted for each other.

[0092] The upper opening of the carrier has a support edge extending outward in the circumferential direction. When the cell culture plug-in 7 is inserted into the cavity of the chip body, the cell culture plug-in 7 is supported on the upper surface of the cavity layer 4 by the support edge.

[0093] This design facilitates the smooth insertion of the cell culture plug-in 7 into the cavity of the chip body and allows for easy removal of the cell culture plug-in 7 from the cavity of the chip body, thus enabling the cell culture plug-in 7 to function as a plug-and-play device. Furthermore, after the cell culture plug-in 7 is inserted into the cavity, the upper clamping plate assembly can be stacked on top of the cavity layer 4. Through the clamping action between the upper clamping plate assembly and the cavity layer 4, a sealed fit is achieved between the lower surface of the support edge of the cell culture plug-in 7 and the upper surface of the cavity layer 4, thereby avoiding the risk of leakage at the connection point between the two in subsequent experimental work.

[0094] It should be noted that the bottom of the containment cavity is not a closed structure. A porous filter membrane structure can be set to separate the cells in the cell culture plug-in 7 from the fluid in the cavity of the chip body. At the same time, the porous filter membrane structure can also be used to culture the endothelial barrier.

[0095] In some embodiments, refer to Figure 3 As shown, the carrier includes a first annular plate 71, a porous filter membrane 72, a second annular plate 73, a third annular plate 74, and a fourth annular plate 76 stacked from bottom to top.

[0096] The porous filter membrane 72 is fixed between the first annular plate 71 and the second annular plate 73. Specifically, the porous filter membrane 72 can be fixed between the first annular plate 71 and the second annular plate 73 by plasma bonding. After the cell culture plug 7 is inserted into the cavity of the chip body, the side of the porous filter membrane 72 corresponds to the microchannel slit 33, that is, the height of the porous filter membrane 72 and the microchannel slit 33 are approximately the same. The porous filter membrane 72 is used to separate the cells in the cell culture plug 7 from the fluid in the cavity. In addition, the porous filter membrane 72 can also be used to culture endothelial barriers, such as the blood-brain barrier, intestinal barrier, and skin barrier.

[0097] In specific implementation, the porous filter membrane 72 can be a polyterephthalic acid porous filter membrane 72, specifically a disc-shaped polyterephthalic acid porous filter membrane 72. The polyterephthalic acid porous filter membrane 72 isolates the cells in the chamber layer 4 from the fluid in the microchannel, avoiding excessive fluid shear stress in the chamber and causing cell damage. At the same time, the polyterephthalic acid porous filter membrane 72 can also be used to culture endothelial cells, etc., to construct endothelial barriers, such as the blood-brain barrier and the intestinal epithelial barrier, so that the constructed RIAE model is more consistent with the in vivo physiological environment.

[0098] The inner wall of the support member has a stepped structure. Specifically, the inner diameter of the third annular plate 74 is larger than that of the second annular plate 73, thereby forming a supporting step between the upper surface of the second annular plate 73 and the bottom of the inner wall of the third annular plate 74, which supports the cell culture scaffold 75. Specifically, the second annular plate 73, the third annular plate 74, and the fourth annular plate 76 together constitute the receiving cavity of the support member. The diameter of the cell culture scaffold 75 is smaller than the inner diameters of the third annular plate 74 and the fourth annular plate 76 but larger than the inner diameter of the second annular plate 73, allowing the cell culture scaffold 75 to be suspended above the porous filter membrane 72 and facilitating its removal from the support member. That is, the cell culture scaffold 75 and the porous filter membrane 72 are separated by the second annular plate 73, without direct contact, to avoid direct contact between the cell culture scaffold 75 and the porous filter membrane 72, which could damage the constructed endothelial barrier.

[0099] The outer diameters of the third annular plate 74, the second annular plate 73, and the first annular plate 71 are smaller than the inner diameter of the cavity 41 of the chamber layer 4, and the outer diameter of the fourth annular plate 76 is larger than the inner diameter of the cavity 41 of the chamber layer 4. The fourth annular plate 76 forms the aforementioned support edge, so that when the cell culture plug-in 7 is inserted into the cavity of the chip body, the portion of the cell culture plug-in 7 located below the fourth annular plate 76 can be accommodated in the cavity of the chip body, and the cell culture plug-in 7 is supported on the upper surface of the chamber layer 4 by the fourth annular plate 76, thereby realizing the plug-and-play function of the cell culture plug-in 7.

[0100] In specific implementations, the first annular plate 71 and the second annular plate 73 can have the same inner and outer diameters. The diameter of the porous filter membrane 72 can be larger than the inner diameters of the first annular plate 71 and the second annular plate 73, but smaller than the outer diameters of the first annular plate 71 and the second annular plate 73. The porous filter membrane 72 is fixed between the second annular plate 73 and the first annular plate 71 using a plasma bonding process. The third annular plate 74 and the fourth annular plate 76 can have the same inner diameter, but the outer diameter of the fourth annular plate 76 is larger than... The outer diameter of the third annular plate 74, the first annular plate 71, the porous filter membrane 72, the second annular plate 73, the third annular plate 74 and the fourth annular plate 76 are stacked from bottom to top. The upper surface of the second annular plate 73, the inner wall of the third annular plate 74 and the inner wall of the fourth annular plate 76 together form a receiving cavity for accommodating the cell culture scaffold 75. The fourth annular plate 76 is formed as a supporting edge extending outward in the circumferential direction from the upper opening of the receiving cavity. The supporting edge supports the upper surface of the chamber layer 4.

[0101] In some embodiments, refer to Figure 1 and Figure 2 As shown, the upper clamping plate assembly includes a first upper clamping plate 6 and a second upper clamping plate 5. The first upper clamping plate 6 is stacked on top of the second upper clamping plate 5. The first upper clamping plate 6 and the second upper clamping plate 5 are respectively provided with two perfusion inlets and outlets 62. Each perfusion inlet and outlet 62 is provided with a perfusion connector 51. The perfusion connector 51 is used to connect an external conduit so as to realize the perfusion of the culture medium through the perfusion connector 51.

[0102] Specifically, two perfusion inlets / outlets 62 can be correspondingly provided on the first upper clamping plate 6 and the second upper clamping plate 5. The two perfusion inlets / outlets 62 can be located on both sides of the two chambers respectively, and each perfusion inlet / outlet 62 is provided with a perfusion connector 51. In use, the culture medium is poured in through the conduit from the perfusion connector 51 of one perfusion inlet / outlet 62 and flows out from the perfusion connector 51 of the other perfusion inlet / outlet 62, thereby realizing the perfusion of the culture medium.

[0103] In specific implementation, the perfusion connector 51 can be a Luer connector. The Luer connector can be connected to an external pump or valve through a conduit to facilitate the perfusion of the culture medium. Under the continuous perfusion of the culture medium, substances between different chambers can be transported through the microchannel layer 3. In this way, the plug-and-play multi-organ chip can more realistically simulate the interaction and signal transmission between different organs in the human body.

[0104] In some embodiments, refer to Figure 1 and Figure 2 As shown, both the first upper clamping plate 6 and the second upper clamping plate 5 are provided with connecting holes 61 that correspond one-to-one with and communicate with the chambers, so as to ensure the air permeability of the cell culture plug 7 inserted into the chamber. In a specific implementation, the surface of the second upper clamping plate 5 facing away from the first upper clamping plate 6 may be provided with a breathable membrane (not shown in the figure), which covers the bottom of the connecting hole 61. Specifically, the breathable membrane can be a breathable but water-permeable membrane, such as a polydimethylsiloxane membrane, to ensure the air permeability of the cell culture plug 7 when culturing cells.

[0105] Furthermore, a flexible membrane is provided between the lower bottom surface of the upper clamping plate assembly and the upper surface of the chamber layer 4. The flexible membrane has a connecting hole corresponding to and communicating with the perfusion hole 42, so that the culture medium poured in from the perfusion connector 51 can enter the perfusion hole 42 through the connecting hole. Specifically, after the cell culture plug 7 is placed in the chamber, it will partially protrude from the upper surface of the chamber layer 4, resulting in a gap between the lower bottom surface of the upper clamping plate assembly and the upper surface of the chamber layer 4. This causes the culture medium poured in from the perfusion connector 51 to easily overflow from the gap. By providing the flexible membrane, it can act as a pad to fill the gap between the lower bottom surface of the upper clamping plate assembly and the upper surface of the chamber layer 4, so that the culture medium poured in from the perfusion connector 51 can smoothly enter the perfusion hole 42.

[0106] Specifically, the thickness of the flexible membrane can be reasonably set based on the distance by which the cell culture insert 7 protrudes from the upper surface of the chamber layer 4. After placing the flexible membrane on the upper surface of the chamber layer 4, it is advisable that the upper surface of the flexible membrane is flush with the upper surface of the cell culture insert 7 to achieve a good spill-proof effect. In addition, if a breathable membrane is provided on the lower bottom surface of the upper clamping plate assembly, the upper surface of the flexible membrane can be set to be flush with the upper surface of the breathable membrane.

[0107] In some embodiments, the materials of the lower clamping plate 1, the first upper clamping plate 6, and the second upper clamping plate 5 can all be polymethyl methacrylate (PMMA). PMMA is chosen as the material for the clamping plates because it is rigid, does not easily deform under pressure, is inexpensive, and is easy to process.

[0108] In some embodiments, the base layer 2, the microchannel layer 3, and the chamber layer 4 can all be made of polydimethylsiloxane; the porous filter membrane 72 can be a circular polyterephthalic acid porous filter membrane 72; the first annular sheet 71, the second annular sheet 73, and the third annular sheet 74 can be made of polydimethylsiloxane; the fourth annular sheet 76 can be made of polydimethylsiloxane soft membrane; and the cell culture scaffold 75 can be a rigid three-dimensional cell culture scaffold.

[0109] In some embodiments, in addition to placing the cell culture scaffold 75, the cavity formed by the carrier of the cell culture plug 7 can also be used to directly culture suspended cells.

[0110] Reference Figure 5 The diagram shown is a flowchart illustrating the multi-organ-on-a-chip fabrication method provided in an embodiment of the present invention.

[0111] Reference Figure 5 As shown, other embodiments of the present invention provide a method for fabricating a multi-organ-on-a-chip, used for fabricating a multi-organ-on-a-chip as described in any of the above embodiments, comprising the following steps:

[0112] S51, fabrication of the chip body;

[0113] S52, Prepare cell culture plug-in 7.

[0114] The main body for chip fabrication includes:

[0115] Prepare an upper clamping plate assembly and a lower clamping plate 1, and provide an irrigation connector 51 on the upper clamping plate assembly;

[0116] Prepare chamber layer 4;

[0117] Microchannel layer 3 and substrate layer 2 were prepared;

[0118] The microchannel layer 3, the base layer 2, and the chamber layer 4 are bonded together so that the chamber layer 4, the microchannel layer 3, and the base layer 2 together form at least two chambers a and b.

[0119] In this embodiment of the invention, by bonding the microchannel layer 3, the substrate layer 2, and the chamber layer 4, the microchannel layer 3, the substrate layer 2, and the chamber layer 4 can be integrated into one unit, which facilitates the subsequent assembly of the chip body and ensures the sealed connection between the microchannel layer 3, the substrate layer 2, and the chamber layer 4, avoiding problems such as leakage during subsequent experiments.

[0120] The following describes in detail, with reference to the accompanying drawings, the methods for preparing the chip body and the cell culture plug-in 7 in the multi-organ chip fabrication method provided in the embodiments of the present invention.

[0121] Reference Figure 6The diagram shown is a schematic representation of the process for preparing the upper clamping plate assembly and the lower clamping plate, and for setting the infusion connector on the upper clamping plate assembly, according to an embodiment of the present invention.

[0122] Reference Figure 6 As shown, in some embodiments, the multi-organ-on-a-chip fabrication method provided by the present invention includes fabricating an upper clamp assembly and a lower clamp 1, and setting an irrigation connector 51 on the upper clamp assembly, comprising:

[0123] S61, process the first upper clamping plate 6, the second upper clamping plate 5, and the lower clamping plate 1.

[0124] Specifically, in this embodiment of the invention, the upper clamping plate assembly may include a first upper clamping plate 6 and a second upper clamping plate 5. Polymethyl methacrylate (PMMA) can be selected as the material for making the clamping plates. Since PMMA is rigid, it is not easily deformed when pressure is applied, and it is inexpensive and easy to process, PMMA can be used as the material for making the first upper clamping plate 6, the second upper clamping plate 5 and the lower clamping plate 1.

[0125] Specifically, the first upper clamping plate 6 and the second upper clamping plate 5 may be provided with infusion inlets and outlets 62 for installing infusion connectors 51; the first upper clamping plate 6 and the second upper clamping plate 5 may also be provided with connecting holes 61 for corresponding to and communicating with the cavities 41 provided in the chamber layer 4 to ensure the air permeability of the multi-organ chip; the first upper clamping plate 6 and the second upper clamping plate 5 may also be provided with multiple docking holes for fastening bolts to pass through, so as to achieve docking and fixing of the first upper clamping plate 6, the second upper clamping plate 5 with the chamber layer 4, the microchannel layer 3, the base layer 2 and the lower clamping plate 1 by fastening bolts and fastening nuts.

[0126] S62, use epoxy resin potting compound to bond the first upper clamping plate 6 and the second upper clamping plate 5.

[0127] Specifically, in this embodiment of the invention, epoxy resin potting compound is used to bond the first upper clamping plate 6 and the second upper clamping plate 5. Here, care should be taken to prevent the glue from clogging the inlet and outlet 62.

[0128] S63, use double-sided tape to fix the irrigation connector 51 at the irrigation inlet / outlet 62 of the first upper clamp 6 and the second upper clamp 5, and use epoxy resin potting compound to seal around the irrigation connector 51.

[0129] Specifically, in this embodiment of the invention, the irrigation connector 51 can be a Luer connector. Polymethyl methacrylate double-sided tape can be used to fix the irrigation connector 51 at the irrigation inlet and outlet 62 of the first upper clamping plate 6 and the second upper clamping plate 5, and epoxy resin potting compound can be used to seal around the connector to prevent liquid from seeping out during the irrigation process.

[0130] S64, using rigid clamps for assistance, pressure is applied by setting fastening bolts and fastening nuts on the first upper clamping plate 6 and the second upper clamping plate 5 to make the adhesion firm, while keeping the first upper clamping plate 6 and the second upper clamping plate 5 tightly fitted. Alternatively, dovetail clamps can be used instead of bolts and fastening nuts to apply pressure.

[0131] Specifically, in this embodiment of the invention, a rigid polymethyl methacrylate clamp is used as an aid, and fastening bolts are inserted through multiple mating holes on the first upper clamping plate 6 and the second upper clamping plate 5, and fastening nuts are screwed onto the fastening bolts to apply pressure to the first upper clamping plate 6 and the second upper clamping plate 5 so that the two are firmly bonded together.

[0132] Reference Figure 7 The diagram shown is a schematic representation of the preparation of the first upper clamping plate, the second upper clamping plate, and the lower clamping plate using the preparation method provided in this embodiment of the invention. It is for illustrative purposes only and is intended to facilitate understanding. Figure 7 (a) shows a physical image of the first upper clamping plate 6 and the second upper clamping plate 5; (b) shows a physical image of the second upper clamping plate 5; (c) shows a physical image of the lower clamping plate 1; (d) shows a physical image of the chip body and the cell culture plug-in 7 assembled together.

[0133] Reference Figure 8 The diagram shown is a schematic flowchart of the process for preparing the chamber layer according to an embodiment of the present invention.

[0134] Reference Figure 8 As shown, in some embodiments, the multi-organ-on-a-chip fabrication method provided by the present invention includes the following steps in fabricating the chamber layer 4:

[0135] S81, polydimethylsiloxane is selected as the material for the chamber layer 4. According to the recommended mixing ratio of polydimethylsiloxane, the curing agent and the main body are mixed and stirred in a preset mass ratio to obtain a polydimethylsiloxane premix.

[0136] Specifically, in this embodiment of the invention, the curing agent and the main body can be mixed and stirred at a mass ratio of 1:10 according to the recommended mixing ratio of polydimethylsiloxane to obtain a polydimethylsiloxane premix.

[0137] S82, pour the polydimethylsiloxane premix of a predetermined thickness into the polystyrene mold.

[0138] Specifically, in this embodiment of the invention, a polydimethylsiloxane premix of a preset thickness can be poured into a polystyrene mold of a first preset volume. For example, a polydimethylsiloxane mixture of about 7 mm thickness can be poured into a polystyrene mold of a size of 130×70×25 mm. The thickness can be controlled by weighing.

[0139] S83, after curing, is demolded to obtain polydimethylsiloxane blocks.

[0140] Specifically, in this embodiment of the invention, a first preset time period can be cured at room temperature, and then a second preset time period can be cured in an oven at a preset temperature. After demolding, a polydimethylsiloxane block with a size of a second preset volume is obtained. For example, curing the first preset time period at room temperature can be 24 hours at room temperature, and curing the second preset time period in an oven at a preset temperature can be 3 hours in an oven at 60°C. After demolding, a polydimethylsiloxane block of 130×70×7mm is obtained.

[0141] S84, using a die cutter of the first preset diameter, a preset number of cavities 41 are cut into the polydimethylsiloxane block to obtain the chamber layer 4.

[0142] Specifically, in this embodiment of the invention, a circular die cutter with a diameter of 35 mm can be used to cut two cavities 41 into the polydimethylsiloxane block to obtain the chamber layer 4. Of course, three or more cavities 41 can also be cut as needed.

[0143] S85, use a polypropylene adhesive sealing film to seal the top of chamber layer 4.

[0144] Specifically, in this embodiment of the invention, the top of the chamber layer 4 is sealed using a polypropylene adhesive sealing film to construct the chamber layer 4.

[0145] Figure 9 This is a schematic diagram of the process for preparing the microchannel layer and substrate layer provided in an embodiment of the present invention.

[0146] Reference Figure 9 As shown, in some embodiments, the multi-organ-on-a-chip fabrication method provided by the present invention includes the following steps in fabricating the microchannel layer 3 and the substrate layer 2:

[0147] S91, using laser engraving technology to cut out channel positive mold patterns from polymethyl methacrylate sheet, thus obtaining a polymethyl methacrylate channel positive mold.

[0148] S92, select a polystyrene mold as the substrate, drip in epoxy resin potting compound, and place a polymethyl methacrylate channel male mold for bonding.

[0149] Specifically, in this embodiment of the invention, a polymethyl methacrylate (PMMA) board is cut to form the channel male mold pattern of the chip; a polystyrene mold of a second preset volume can be selected as the substrate. For example, a polystyrene mold of 130×70×25mm is selected as the substrate, epoxy resin potting compound is dripped in, and the PMMA channel male mold is placed on it for pasting.

[0150] S93, after curing, yields a microchannel layer mold and a substrate layer mold.

[0151] Specifically, in this embodiment of the invention, the material can first be cured at room temperature for a third preset time period, and then cured in an oven at a preset temperature for a fourth preset time period. For example, the third preset time period can be cured at room temperature for 24 hours, and the fourth preset time period can be cured in an oven at 60°C for 3 hours, to obtain the microchannel layer mold and the substrate layer mold.

[0152] S94, polydimethylsiloxane is selected as the material for the microchannel layer 3 and the base layer 2. The polydimethylsiloxane premix, which is prepared by mixing the curing agent and the main body in a preset mass ratio, is poured into the microchannel layer mold and the base layer mold, so that the polydimethylsiloxane is at least at a preset distance above the top of the polymethyl methacrylate channel positive mold.

[0153] Specifically, in this embodiment of the invention, the curing agent and the main body can be mixed and stirred at a mass ratio of 1:10 according to the recommended mixing ratio of polydimethylsiloxane to obtain a polydimethylsiloxane premix. The polydimethylsiloxane premix is ​​then poured into the microchannel layer mold and the base layer mold, so that the polydimethylsiloxane is at least 1 mm above the top of the polymethyl methacrylate channel positive mold.

[0154] S95, after curing and demolding, yields microchannel layer 3 and base layer 2.

[0155] Specifically, in this embodiment of the invention, a fifth preset time period can be cured at room temperature, and then a sixth preset time period can be cured in an oven at a preset temperature. For example, curing the fifth preset time period at room temperature can be curing for 24 hours at room temperature, and curing the sixth preset time period in an oven at a preset temperature can be curing for 3 hours in an oven at 60°C. After demolding, the microchannel layer 3 and the substrate layer 2 of the polydimethylsiloxane chip that replicates the channel positive mold structure are prepared.

[0156] It should be understood that, based on experience in constructing polymethyl methacrylate (PMMA) chips using laser engraving technology, polydimethylsiloxane (PDMS) premixes do not exhibit good compatibility with PMMA, epoxy resin potting compounds, or polystyrene culture dishes. PMS premixes can be easily demolded from these three materials. However, after oxygen plasma hydrophilic treatment, PMMA and polystyrene culture dishes can be firmly bonded together using epoxy resin potting compounds. Meanwhile, polymethyl methacrylate (PMMA) can be easily fabricated into chips with high aspect ratios using laser engraving technology. Therefore, this invention combines high aspect ratio PMMA laser engraving technology. By using epoxy resin potting compound to bond oxygen plasma-treated polystyrene culture dishes and laser-engraved PMMA (1-5 mm thick), microchannel layer 3 and substrate layer molds with different aspect ratios are constructed. Then, microchannel layer 3 and substrate layer 2 are fabricated using microchannel layer 3 and substrate layer molds, and microchannel layer 3 and substrate layer 2 are bonded to glass. The biocompatibility of the chip is evaluated to establish a laser engraving-based mold fabrication process.

[0157] Reference Figure 10 The diagram shown is a schematic flowchart of the preparation of a cell culture plug-in according to an embodiment of the present invention. (Refer to...) Figure 11 The diagram shown is a schematic representation of the processing steps of the cell culture plug-in provided in an embodiment of the present invention. It is for illustrative purposes only and is intended to facilitate understanding. Figure 11 (a) shows die-cutting blades of different diameters; (b) shows two polydimethylsiloxane discs (i.e., the first annular disc 71 and the second annular disc 73) with a thickness of 1 mm and an inner diameter of 18 mm; (c) shows a polyterephthalic acid membrane with a diameter of 25 mm and a pore size of 0.45 μm placed in the center of one of the polydimethylsiloxane discs; (d) shows the polydimethylsiloxane disc with the polyterephthalic acid membrane sandwiched between the bonded discs on the left and the insert chamber layer 4 (i.e., the third annular disc 74) with a thickness of 4 mm and an inner diameter of 22 mm to be bonded on the right; (e) shows the insert chamber with a thickness of 1 mm and a diameter of 45 mm to be bonded on the left and the polydimethylsiloxane (i.e., the fourth annular disc 76) to be bonded for connection with the chip body on the right; (f) shows a physical image of the cell culture insert 7.

[0158] Reference Figure 10 As shown, in some embodiments, the preparation of the cell culture plug-in 7 in the multi-organ-on-a-chip fabrication method provided by the present invention includes:

[0159] S101, using a petri dish to prepare three polydimethylsiloxane discs with a first preset thickness and one with a second preset thickness.

[0160] Specifically, in this embodiment of the invention, three 1mm thick and one 4mm thick polydimethylsiloxane discs can be made using a petri dish.

[0161] S102, using die-cutting blades with second and third preset diameters on two polydimethylsiloxane discs of first preset thickness to cut out rings, wherein the second preset diameter is larger than the third preset diameter.

[0162] Specifically, in this embodiment of the invention, a ring can be cut out using die-cutting blades with diameters of 32 mm and 18 mm on a polydimethylsiloxane disc with a thickness of 1 mm, to obtain... Figure 11 The first annular piece 71 and the second annular piece 73 are shown in (b) of the diagram.

[0163] S103, a polyterephthalic acid porous filter membrane 72 with a diameter of a first preset length and a pore size of a second preset length is attached to the center of one of the polydimethylsiloxane discs with a first preset thickness. The two polydimethylsiloxane discs are then bonded together by plasma bonding, so that the porous filter membrane 72 is fixed between the two polydimethylsiloxane discs with the first preset thickness. The material of the porous filter membrane 72 includes at least one of polyethylene terephthalate, polycarbonate, polyester, polyimide, polyvinylidene fluoride, and polydimethylsiloxane.

[0164] Specifically, in this embodiment of the invention, a polyethylene terephthalate (PET) film with a diameter of 25 mm and a pore size of 0.45 micrometers can be adhered to the center of one of the polydimethylsiloxane (PDS) ring sheets to obtain the desired result. Figure 11 As shown on the left side of (c), the two polydimethylsiloxane ring sheets are then bonded together by plasma bonding. This step allows the porous filter membrane 72 to be flatly fixed between the two polydimethylsiloxane ring sheets. Figure 11 As shown on the left side of (d) in the diagram.

[0165] S104, a ring is cut out using die-cutting blades with a second and a fourth preset diameter on a polydimethylsiloxane disc of a second preset thickness, and a disc is cut out using a die-cutting blade with a fifth preset diameter on another polydimethylsiloxane disc of a first preset thickness, and then plasma bonded sequentially, wherein the second preset diameter is greater than the fourth preset diameter, the fourth preset diameter is greater than the third preset diameter, and the fifth preset diameter is greater than the second preset diameter.

[0166] Specifically, in this embodiment of the invention, a ring-shaped sheet can be obtained by using die-cutting blades with diameters of 32 mm and 22 mm on a polydimethylsiloxane sheet with a thickness of 4 mm. Figure 11 As shown on the right side of (d) in the image, a circular sheet is obtained by using a die-cutting tool with a diameter of 45 mm on a 1 mm thick polydimethylsiloxane. Figure 11 As shown on the right side of (e) in the diagram, they are sequentially plasma bonded.

[0167] S105. Use a scalpel, die cutter, or punch to cut a hole in the center of the top polydimethylsiloxane disc to serve as the entry point for the cell culture scaffold 75, thus completing the processing of the cell culture insert 7.

[0168] Specifically, in this embodiment of the invention, a hole is cut in the center of the top polydimethylsiloxane disc using a cutting tool to serve as the placement entrance for the rigid three-dimensional cell culture scaffold 75. Figure 11 As shown in (f), the processing of cell culture plugin 7 can be completed.

[0169] In some embodiments of the present invention, the multi-organ-on-a-chip fabrication method further includes:

[0170] Multiple docking holes 11 are provided on the lower clamping plate 1, the base layer 2, the microchannel layer 3, the chamber layer 4, and the upper clamping plate assembly. The docking is fixed by inserting fastening bolts into the multiple docking holes 11 and tightening nuts on the fastening bolts.

[0171] Specifically, in this embodiment of the invention, multiple docking holes 11 are provided on the first upper clamping plate 6, the second upper clamping plate 5, the lower clamping plate 1, the chamber layer 4, the microchannel layer 3 and the base layer 2. By using fastening bolts and fastening nuts to dock and fix the multiple docking holes 11 respectively, the chip body and the cell culture plug-in 7 can be assembled into one unit to obtain a breathable, transparent, detachable plug-and-play multi-organ chip.

[0172] Reference Figure 12 As shown, some embodiments of the present invention provide a multi-organ-on-a-chip application method, employing a multi-organ-on-a-chip as described in any of the above embodiments, including the following steps:

[0173] S121, cells are cultured in N cell culture plug-ins 7 respectively, of which 1 to N-1 cell culture plug-ins 7 are irradiated and cultured as irradiated cells, and the remaining cell culture plug-ins 7 are not irradiated and cultured as unirradiated cells, N≥2;

[0174] S122, All cell culture plug-ins 7 are assembled into the corresponding chambers of the multi-organ chip, wherein the cell culture plug-in 7 containing irradiated cells is used as the upstream cell culture plug-in in the perfusion process, and the cell culture plug-in 7 containing unirradiated cells is used as the downstream cell culture plug-in in the perfusion process.

[0175] S123, culture medium is infused into the multi-organ chip through the perfusion connector 51, with a preset perfusion duration to simulate fluid flow and cell interaction in the in vivo environment.

[0176] With this setup, cells are cultured in N cell culture modules 7, and 1 to N-1 of these modules 7 are irradiated to cultivate irradiated cells, while the remaining modules are not irradiated to cultivate unirradiated cells. This allows for the separate acquisition of irradiated and unirradiated cells. All cell culture modules 7 are then assembled into the corresponding chambers of a multi-organ chip. By perfusing the multi-organ chip, the flow of fluids and cell interactions in the in vivo environment are simulated. In this way, a partial cell radiation model can be constructed without the use of a radiation shielding device, making the operation simple and convenient.

[0177] It should be noted that the types of cells cultured in different cell culture plugins 7 can be different.

[0178] It should be noted that, in order to study the peripheral cell separation effect caused by irradiated cells, the cell culture plug 7 containing irradiated cells was used as the upstream cell culture plug during the perfusion process, while the cell culture plug 7 containing unirradiated cells was used as the downstream cell culture plug during the perfusion process.

[0179] Furthermore, culturing irradiated cells in the cell culture plug-in 7 can specifically include: constructing the required endothelial barrier, such as the blood-brain barrier, intestinal barrier, or skin barrier, within the carrier of the cell culture plug-in 7; simultaneously, culturing the required cells in the cell culture scaffold 75 of the cell culture plug-in 7, irradiating the cell culture scaffold 75 containing the required cells, and then placing it back into the carrier of the cell culture plug-in 7 after irradiation. Of course, in specific implementations, the step of constructing the endothelial barrier within the carrier of the cell culture plug-in 7 can be omitted as needed. Additionally, both the carrier with the constructed endothelial barrier and the cell culture scaffold 75 containing the cells can be irradiated as needed.

[0180] The specific steps for culturing unirradiated cells in cell culture plug-in 7 are largely the same as those for culturing irradiated cells in cell culture plug-in 7, the difference being that the irradiation treatment step is omitted. Furthermore, in practice, it is optional to construct an endothelial barrier within the carrier of cell culture plug-in 7, depending on the needs.

[0181] It should be noted that, in specific implementations, the multi-organ-on-a-chip can have two chambers, and correspondingly, two cell culture inserts 7. The two cell culture inserts 7 can be detachably inserted into the two chambers in a one-to-one correspondence. When using this multi-organ-on-a-chip to culture cells and conduct research, cells can be cultured separately in the two cell culture inserts 7. One cell culture insert 7 is irradiated to culture irradiated cells, while the other cell culture insert 7 is not irradiated to culture unirradiated cells.

[0182] Of course, the number of chambers constructed in a multi-organ-chip can be three or more, and correspondingly, the number of cell culture inserts 7 can also be three or more. Multiple cell culture inserts 7 can be detachably inserted into multiple chambers in a one-to-one correspondence. When using this multi-organ-chip to culture cells and conduct research, cells can be cultured separately in all cell culture inserts 7, or, as needed, only in a portion of the cell culture inserts 7. Cell culture inserts 7 without cultured cells can be placed in their corresponding chambers, or, as needed, membranes can be placed over the corresponding chambers to replace the cell culture inserts. This increases the applicability of the multi-organ-chip, allowing it to be used not only to study interactions between two types of cells, but also to study interactions between three or more types of cells.

[0183] The following section describes the plug-and-play multi-organ chip provided by the present invention with reference to a specific embodiment.

[0184] To investigate the peripheral mononuclear cell-mediated irradiation-induced peripheral intracellular echogenic encephalopathy (RIAE) caused by radiation damage to the central nervous system (CNS), an in vitro model was constructed using a plug-and-play multi-organ microarray, which is briefly described below:

[0185] A blood-brain barrier (BBB) ​​was constructed on a polyethylene terephthalate (PET) membrane coated with GelMA (gelatinmethacryloyl) within the upstream cell culture module. Simultaneously, human astrocytoma cell line U-87MG and human neuroblastoma cell line SH-SY5Y were co-cultured on the 3D cell culture scaffold. To simulate radiation-induced CNS (central nervous system) damage, the scaffold was irradiated with 15 Gy at a rate of 1 Gy / min. After irradiation, the scaffold was returned to the upstream module chamber and placed on the BBB layer. The downstream cell culture module was seeded with the human acute monocytic leukemia (THP-1) cell line as an in vitro model of peripheral mononuclear cells. Subsequently, the CNS module and the mononuclear cell module were assembled into a plug-and-play multi-organ chip and continuously perfused for 48 hours to simulate fluid flow and cell interactions in the in vivo environment. Forty-eight hours later, key indicators were detected and analyzed, including morphological observation of the upstream BBB layer, TEER (Transepithelial Electric Resistance) measurement, sodium fluorescein permeability detection, THP-1 adherent cell number analysis, cell viability detection, and related gene transcription levels. The control group chip underwent the same treatment as the experimental group, except that the CNS plug was not irradiated.

[0186] Figure 13 This is a schematic diagram showing the TEER value measurement results of BBB 48 hours after perfusion, as provided in an embodiment of the present invention.

[0187] Transendothelial resistance (TEER) was measured in the brain microvascular endothelial cell line (bBB) to evaluate the degree of tight junction formation in the hCMEC / D3 cell line. TEER is an important indicator of the degree of tight junctions between cell layers; a higher TEER value indicates stronger tight junctions between cell layers. Results are as follows: Figure 13 As shown, data from parallel treatments within a group were analyzed after subtracting the standard deviation from the mean, and the standard deviation was marked on the graph as error bars. Statistical analysis between groups was performed using t-tests or ANOVA, and significant differences were expressed as follows: *** represents p < 0.001. The number of replicates in all experiments was n ≥ 3. The TEER value of the control group was approximately (509 ± 25) Ω·cm. 2 The TEER value of the irradiated group was approximately (224±31) Ω·cm. 2 The results showed that the TEER value in the irradiated group was significantly lower than that in the control group, suggesting that radiation damage to nerve cells induces increased BBB permeability.

[0188] To investigate the effects of radiation-induced nerve cell damage on peripheral mononuclear cells, a neural-monocyte RIAE model was constructed using a plug-and-play multi-organ chip, and multiple tests were performed on THP-1 cells in the downstream plug-in of the chip.

[0189] Figure 14 This is a schematic diagram illustrating the analysis results of THP-1 adhesion degree and cell viability provided in an embodiment of the present invention.

[0190] Figure 14 (a) indicates THP-1 cell staining. Figure 2 Before and after value-based processing; Figure 14 (b) indicates that the area attached to the wall was calculated using the binarized image (****p<0.0001); Figure 14 (c) indicates the cell viability of THP-1 as determined by the CCK8 (Cell Counting Kit-8) method (***p<0.001).

[0191] First, the cell viability of THP-1 cells within the downstream plug-in was evaluated using the CCK8 assay. The results are as follows: Figure 14 As shown in c, the survival rate of THP-1 cells in the irradiated group was approximately 90% of that in the control group, indicating that the proliferation of THP-1 cells in the irradiated group was inhibited.

[0192] Secondly, the THP-1 cells that had adhered and differentiated on the downstream terephthalic acid (PET) membrane were stained with Giemsa stain for observation under an optical microscope. The results showed that the THP-1 cells on the PET membrane in the control group were sparsely distributed, while the THP-1 cells in the irradiated group showed obvious clustering. Furthermore, the adherent area of ​​the THP-1 cells in the irradiated group was significantly higher than that in the control group. Figure 14 a and Figure 14 b). The above results indicate that the THP-1 cells in the irradiated group exhibited a higher degree of adherence and differentiation.

[0193] Figure 15 This is a schematic diagram illustrating the analysis results of relative RNA expression levels in THP-1 cells provided in an embodiment of the present invention.

[0194] Specifically, in combination Figure 15 Irradiated neural cells were cultured in the upstream plug-in chamber of the chip. After 48 hours of perfusion culture, THP-1 RNA was extracted and analyzed for CD14 (Cluster of differentiation 14), CXCL-1 (Chemokine (CXC motif) ligand 1), CCL-20 (Chemokine (CC motif) ligand 20), and TNF-α (Tumor) ligands. The relative expression levels of the genes Necrosis Factor-α, IL-6 (Interleukin-6), and TLR-2 (Toll-like Receptor-2) were analyzed (data from parallel samples within each group were analyzed after taking the mean plus or minus the standard deviation, and the standard deviation was marked on the graph as error bars; statistical analysis between samples was performed using t-tests or ANOVA, and significant differences were expressed as follows: n≥3ns - no significant difference, **p<0.01, ***p<0.001, ****p<0.0001. Number of replicates in all experiments).

[0195] In addition, changes in the transcriptional levels of several cytokines and monocyte activation markers in THP-1 were detected, combined with... Figure 15 It includes CD14, CXCL-1, CCL-20, TNF-α, IL-6, and TLR-2.

[0196] CD14 is a surface marker of monocytes and plays an important role in monocyte differentiation and activation. Therefore, upregulation of CD14 may indicate that THP-1 cells are differentiating into monocytes / macrophages or dendritic cells. This finding further supports the effects of irradiated neurons on THP-1 cells.

[0197] The expression levels of pro-inflammatory cytokines TNF-α and IL-6 were upregulated in THP-1 cells, with a particularly significant increase in IL-6 RNA levels. IL-6 is an important inflammatory mediator, and its upregulation may indicate that cells are in an inflammatory state or have been stimulated by inflammation. This result further supports the regulation of the inflammatory response of irradiated neurons in THP-1 cells.

[0198] This embodiment also observed upregulation of the expression levels of chemokines CXCL-1 and CCL-20 in THP-1 cells. CXCL-1 and CCL-20 are important chemokines that can attract specific cell types to migrate to specific tissues or sites. This finding suggests that irradiating nerve cells may trigger a chemotactic response in THP-1 cells.

[0199] The expression of TLR-2 in THP-1 cells was also upregulated. TLR-2 is an important immune receptor involved in cellular recognition of bacteria, viruses, and other pathogens and in immune responses. This result suggests that irradiated nerve cells may influence the immune response by regulating TLR-2 expression in THP-1 cells.

[0200] In summary, radiation-damaged nerve cells can induce mononuclear cell inflammation.

[0201] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0202] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A multi-organ chip, characterized in that, Includes the chip body and at least two cell culture plugs (7); The chip body includes a lower clamping plate (1), a base layer (2), a microchannel layer (3), a chamber layer (4), and an upper clamping plate assembly stacked from bottom to top; The chamber layer (4) is provided with at least two cavities (41) spaced apart. The microchannel layer (3) is provided with microchannel holes (31) that correspond one-to-one with and communicate with the cavities (41). The base layer (2) covers the bottom of the microchannel layer (3). The chamber layer (4), the microchannel layer (3) and the base layer (2) together form at least two chambers (a, b). Each chamber (a, b) can accommodate one cell culture plug (7). The cell culture plug (7) is detachably inserted into the chamber (a, b). The upper clamping plate assembly is provided with communicating holes (61) that correspond one-to-one with and communicate with the chambers (a, b). The upper clamping plate assembly is provided with an irrigation connector (51), the chamber layer (4) is provided with an irrigation hole (42) corresponding to and communicating with the irrigation connector (51), the microchannel layer (3) is provided with an irrigation slit (32) corresponding to and communicating with the irrigation hole (42), the irrigation slit (32) is communicating with the microchannel hole (31), and the microchannel layer (3) is also provided with a microchannel slit (33) communicating with two adjacent chambers (a, b).

2. The multi-organ chip according to claim 1, characterized in that, The lower clamping plate (1), the base layer (2), the microchannel layer (3), the chamber layer (4) and the upper clamping plate assembly are all provided with multiple docking holes (11) so that fastening bolts are inserted into the multiple docking holes (11) respectively, and fastening nuts are screwed onto the fastening bolts for docking and fixing. And / or, a flexible membrane is provided between the bottom surface of the upper clamping plate assembly and the upper surface of the chamber layer (4), and a connecting hole corresponding to and communicating with the irrigation hole (42) is provided on the flexible membrane so that the culture medium injected from the irrigation connector (51) enters the irrigation hole (42) through the connecting hole; And / or, the upper clamping plate assembly includes a first upper clamping plate (6) and a second upper clamping plate (5), the first upper clamping plate (6) is stacked on top of the second upper clamping plate (5), the first upper clamping plate (6) and the second upper clamping plate (5) are respectively provided with two perfusion inlets and outlets (62), each perfusion inlet and outlet (62) is provided with a perfusion connector (51), the perfusion connector (51) is used to connect an external conduit so as to realize the perfusion of culture medium through the perfusion connector (51).

3. The multi-organ chip according to claim 1, characterized in that, The cell culture insert (7) includes a carrier and a cell culture scaffold (75); The carrier has a receiving cavity with an opening at the top, and the cell culture scaffold (75) is placed in the receiving cavity. The cell culture scaffold (75) is used to culture adherent cells and construct an in vitro model of a physiological system. The upper opening of the carrier has a support edge extending outward in the circumferential direction, and the cell culture plug (7) is supported on the upper surface of the chamber layer (4) by the support edge.

4. The multi-organ chip according to claim 3, characterized in that, The carrier includes a first annular sheet (71), a porous filter membrane (72), a second annular sheet (73), a third annular sheet (74), and a fourth annular sheet (76) stacked from bottom to top; The porous filter membrane (72) is fixed between the first annular plate (71) and the second annular plate (73). After the cell culture plug (7) is inserted into the chambers (a, b), the side of the porous filter membrane (72) corresponds to the microchannel slit (33) and is used to separate the cells in the cell culture plug (7) from the fluid in the chambers (a, b) and to construct the endothelial barrier. The second annular plate (73), the third annular plate (74), and the fourth annular plate (76) together form the receiving cavity. The diameter of the cell culture scaffold (75) is smaller than the inner diameter of the third annular plate (74) and the fourth annular plate (76) but larger than the inner diameter of the second annular plate (73), so that the cell culture scaffold (75) can be suspended above the porous filter membrane (72). The outer diameters of the third annular plate (74), the second annular plate (73), and the first annular plate (71) are smaller than the inner diameter of the cavity (41) of the chamber layer (4), and the outer diameter of the fourth annular plate (76) is larger than the inner diameter of the cavity (41) of the chamber layer (4). The fourth annular plate (76) forms the supporting edge, and the cell culture plug (7) is supported on the upper surface of the chamber layer (4) by the fourth annular plate (76).

5. A method for fabricating a multi-organ-on-a-chip, characterized in that, The preparation of the multi-organ chip as described in any one of claims 1 to 4 includes the following steps: Fabrication of the chip substrate; Prepare cell culture plug-in (7); The chip preparation body includes: Prepare an upper clamping plate assembly and a lower clamping plate (1), and provide an irrigation connector (51) on the upper clamping plate assembly; Prepare the chamber layer (4); Microchannel layer (3) and substrate layer (2) were prepared; The microchannel layer (3), the basal layer (2), and the chamber layer (4) are bonded together so that the chamber layer (4), the microchannel layer (3), and the basal layer (2) together form at least two chambers (a, b).

6. The method for fabricating a multi-organ-on-a-chip according to claim 5, characterized in that, The preparation of the upper clamping plate assembly and the lower clamping plate (1), and the provision of the infusion connector (51) on the upper clamping plate assembly, includes: Process the first upper clamping plate (6), the second upper clamping plate (5), and the lower clamping plate (1); The first upper clamping plate (6) and the second upper clamping plate (5) are bonded together using epoxy resin potting compound; Use double-sided tape to fix the injection connector (51) at the injection inlet and outlet (62) of the first upper clamp (6) and the second upper clamp (5), and use epoxy resin potting compound to seal around the injection connector (51). Using rigid clamps as an aid, pressure is applied by setting fastening bolts and fastening nuts on the first upper clamping plate (6) and the second upper clamping plate (5) to make the adhesion firm.

7. The method for fabricating a multi-organ-on-a-chip according to claim 5, characterized in that, The preparation of the chamber layer (4) includes: Polydimethylsiloxane was selected as the material for the chamber layer (4). According to the recommended mixing ratio of polydimethylsiloxane, the curing agent and the main body were mixed and stirred in a preset mass ratio to obtain a polydimethylsiloxane premix. Pour a premix of polydimethylsiloxane of a predetermined thickness into a polystyrene mold; After curing, the polydimethylsiloxane block is obtained by demolding. Using a die cutter of the first preset diameter, a preset number of cavities (41) are cut into the polydimethylsiloxane block to obtain a chamber layer (4); The top of the chamber layer (4) is sealed using a polypropylene adhesive sealing film.

8. The method for fabricating a multi-organ-on-a-chip according to claim 5, characterized in that, The preparation of the microchannel layer (3) and the substrate layer (2) includes: Using laser engraving technology, a channel positive mold pattern is cut out from a polymethyl methacrylate sheet to obtain a polymethyl methacrylate channel positive mold. A polystyrene mold was selected as the substrate, epoxy resin potting compound was dripped in, and the polymethyl methacrylate channel positive mold was placed on top and pasted. After curing, a microchannel layer mold and a substrate layer mold are obtained; Polydimethylsiloxane was selected as the material for the microchannel layer (3) and the base layer (2). The polydimethylsiloxane premix, which was prepared by the curing agent and the main body in a preset mass ratio, was poured into the microchannel layer mold and the base layer mold, so that the polydimethylsiloxane was at least higher than the top of the polymethyl methacrylate channel positive mold by a preset distance. After curing, the microchannel layer (3) and the base layer (2) are obtained by demolding.

9. The method for fabricating a multi-organ-on-a-chip according to claim 5, characterized in that, The preparation of the cell culture plug (7) includes: Three polydimethylsiloxane discs with a first preset thickness and one with a second preset thickness were prepared using a petri dish; Using die-cutting blades of a second and a third preset diameter on two polydimethylsiloxane discs of a first preset thickness, rings are cut out; A porous filter membrane with a diameter of a first preset length and a pore size of a second preset length is attached to the center of one of the polydimethylsiloxane discs with a first preset thickness. The two polydimethylsiloxane discs are then bonded together by plasma bonding, thereby fixing the porous filter membrane between the two polydimethylsiloxane discs with the first preset thickness. The material of the porous filter membrane includes at least one of polyethylene terephthalate, polycarbonate, polyester, polyimide, polyvinylidene fluoride, and polydimethylsiloxane. A ring is cut out using a die-cutting blade with a second preset diameter and a fourth preset diameter on a polydimethylsiloxane disc of a second preset thickness. A disc is cut out using a die-cutting blade with a fifth preset diameter on another polydimethylsiloxane disc of a first preset thickness. The discs are then plasma bonded sequentially. The second preset diameter is greater than the fourth preset diameter, the fourth preset diameter is greater than the third preset diameter, and the fifth preset diameter is greater than the second preset diameter. Use a sculptor, die cutter, or punch to cut a hole in the center of the top polydimethylsiloxane disc to serve as an entry point for the cell culture scaffold, thus completing the processing of the cell culture insert (7).

10. A method for applying a multi-organ-on-a-chip, characterized in that, The multi-organ chip as described in any one of claims 1 to 4 includes the following steps: Cells are cultured in N cell culture plugs (7), of which 1 to N-1 cell culture plugs (7) are irradiated and cultured as irradiated cells, while the remaining cell culture plugs (7) are not irradiated and cultured as unirradiated cells, N≥2; All cell culture plugs (7) are assembled into the corresponding chambers (a, b) of the multi-organ chip, wherein the cell culture plug (7) containing irradiated cells is used as the upstream cell culture plug in the perfusion process, and the cell culture plug (7) containing unirradiated cells is used as the downstream cell culture plug in the perfusion process. Culture medium is infused into the multi-organ chip through the perfusion connector (51) for a preset perfusion duration to simulate fluid flow and cell interaction in the in vivo environment.

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