Microfluidic cartridge for automated perfusion culture of cells

Abstract

The application discloses a micro-fluidic card box for cell automated perfusion culture, and belongs to the field of bioengineering and automatic control, which comprises a substrate, a card box body and a cover plate; the card box body comprises at least one culture chamber, and a cell filter screen is arranged in the culture chamber; the culture chamber comprises a perfusion inlet and a perfusion outlet connected thereto, and the perfusion inlet is higher than the perfusion outlet; the card box body further comprises a liquid one-way valve assembly, an internally arranged perfusion pipeline and an outlet pipeline, the perfusion pipeline is higher than the outlet pipeline, and the liquid one-way valve assembly limits the one-way flow of a culture medium; a plurality of sensors for monitoring cell perfusion culture related process parameters are fixed on the substrate. The micro-fluidic card box for cell automated perfusion culture aims to provide cell automated perfusion culture function, continuously monitor and regulate the cell perfusion culture environment. The micro-fluidic card box can improve cell culture throughput, shorten fermentation time and reduce the volume of the cell culture system.

Description

Technical Field

[0001] This application relates to the fields of bioengineering and automatic control, and more specifically, to a microfluidic cartridge for automated cell perfusion culture. Background Technology

[0002] Molecular biology and gene editing technologies have had a profound impact on the bioengineering industry. Many industrial applications of these technologies benefit from the large-scale culture and synthesis capabilities of cell systems such as bacteria, yeast, fungi, and insects. Traditional cell scale-up culture begins with a given cell line, typically involving initial screening in a large number of microtiter plates, followed by secondary screening in shake flasks to select the best-performing candidate cell lines. Further process development is then conducted under laboratory-scale reactor conditions, culminating in process validation and pilot-scale experiments. The bioreactors involved in this process vary by orders of magnitude in size, such as microplates (microliters to milliliters), shake flasks (100-1000 mL), laboratory fermenters (1-50 L), pilot-scale (300-1000 L), and factory-scale (2-500 cubic meters). Therefore, industrial scale-up requires optimizing conditions based on production volume and screening for specific cell lines, such as pre-screening in shake flasks or laboratory fermenters to tolerate subsequent high-salt, high-temperature, high-acid / high-alkali, and high-product-accumulation environments. This process involves numerous parallel control experiments.

[0003] Existing microbioreactors, mainly consisting of microplates and shake flasks, can perform batch culture, which can be completed manually or periodically by pipetting. Microplate culture has a long growth cycle and low biosynthetic yield. Shakers can shorten fermentation time, but cannot monitor parameters in real time. Recently, parallel bioreactors (working volume around 250 mL) have emerged, comparable in size to shake flasks, possessing parameter acquisition and control functions equivalent to laboratory-scale reactors. They can perform batch and fed-batch culture, but their large culture volume makes high throughput difficult. To address this, Sartorius recently launched the internationally leading, high-throughput microbioreactor ambr, whose culture conditions are similar to large-scale fermentation systems. It consists of 12 or 24 single-use bioreactor systems operating in parallel, with a culture volume of 10–15 mL. While automated liquid handling, including sampling and replenishment of various liquids, can be performed by a robotic arm, the replenishment time is too long, approximately one hour for one cycle of liquid handling across 24 wells. Furthermore, all reactors require integrated temperature control and cannot be individually controlled; and their control parameters cannot be modified during operation. Therefore, the demand for automated perfusion culture in cell microreactors has not yet been fully met.

[0004] In addition, new demands have emerged in this field for cell cluster culture, such as organ-on-a-chip and organoids. These bioengineered cells are three-dimensional, self-renewing cell populations that have differentiated or are about to differentiate into multiple organ-specific cell types, providing a highly physiologically relevant miniature simulated organ. Existing three-dimensional culture methods for organ-on-a-chip and organoids mostly use well plates. Although this method allows cells to survive and grow in the culture medium, the static culture method of well plates, where the culture medium does not circulate, makes it difficult for cell metabolic products to be removed, and lacks precise control over the addition of culture medium, resulting in a very low cell survival rate. Summary of the Invention

[0005] This application provides a microfluidic cartridge for automated cell perfusion culture, which aims to provide cell perfusion culture function, complete cell line culture for multiple growth cycles, and realistically simulate the continuous blood supply environment in the cell, reconstruct the physiological environment of cell lines or cell populations in vivo, thereby increasing cell culture throughput, shortening fermentation time and reducing the volume of the culture system.

[0006] This application provides a microfluidic cartridge for automated cell perfusion culture, comprising:

[0007] Substrate, card holder body and cover;

[0008] The card holder body is disposed on the substrate, and the cover plate is disposed on the side of the card holder body opposite to the substrate;

[0009] The cartridge body includes at least one culture chamber, the culture chamber extends through the cartridge body in a first direction, and a cell filter is disposed inside the culture chamber;

[0010] The culture chamber includes an inlet and an outlet, and the inlet and outlet are connected to the culture chamber.

[0011] The main body of the card box also includes a side-mounted irrigation fluid inlet and an irrigation fluid outlet, an internal irrigation pipe and an outlet pipe, the irrigation inlet and the irrigation fluid inlet are connected by the irrigation pipe, and the irrigation outlet and the irrigation fluid outlet are connected by the outlet pipe;

[0012] In the first direction, the irrigation outlet is closer to the substrate than the irrigation inlet, and the outlet pipe is closer to the substrate than the irrigation pipe; the first direction is perpendicular to the substrate.

[0013] Optionally, an irrigation downbend pipe is provided between the irrigation inlet and the irrigation pipe, with one end of the irrigation downbend pipe connected to the irrigation inlet and the other end connected to the irrigation pipe;

[0014] An irrigation upbend pipe is provided between the irrigation outlet and the outlet pipe. One end of the irrigation upbend pipe is connected to the irrigation outlet, and the other end is connected to the outlet pipe.

[0015] Optionally, multiple culture chambers are provided, and the multiple culture chambers are arrayed on the cartridge body;

[0016] The perfusion fluid inlet is provided in multiple ways, and each of the multiple perfusion fluid inlets corresponds one-to-one with the perfusion inlet of the multiple culture chambers; the perfusion pipe is provided in multiple ways, and each of the multiple perfusion pipes corresponds one-to-one with the perfusion inlet of the multiple culture chambers.

[0017] Multiple perfusion fluid outlets and multiple outlet pipes are provided, with each perfusion fluid outlet corresponding to one of the multiple outlet pipes; the perfusion outlets of multiple culture chambers located in the same column or row are connected to one of the multiple outlet pipes.

[0018] Optionally, the cell filter is configured as a cup-shaped structure with one end open, and at least one filter is provided on the side wall or bottom wall away from the opening of the cup-shaped structure;

[0019] The pore size of the filter is smaller than the diameter of the target cultured cells inside the cell filter.

[0020] Optionally, the microfluidic cartridge further includes a liquid one-way valve assembly, which is connected to the perfusion fluid inlet;

[0021] The liquid one-way valve assembly includes a one-way filter membrane, the side of the one-way filter membrane facing the perfusion fluid inlet is the first side, and the side of the one-way filter membrane away from the perfusion fluid inlet is the second side.

[0022] Specifically, when liquid permeates the unidirectional filter membrane, the flow resistance from the second side to the first side is less than the flow resistance from the first side to the second side.

[0023] Optionally, the substrate is provided with at least one closed sealing groove, the culture chamber is located in the corresponding sealing groove, and a sealing ring is provided in the sealing groove.

[0024] Optionally, at least one set of fasteners is provided on the substrate, the fasteners including a plurality of screws, the screws being disposed through the substrate, and the plurality of screws being located outside the sealing groove;

[0025] The card holder body is embedded with a magnetic tube that corresponds one-to-one with the screws. The screws can be inserted into the center of the magnetic tubes to achieve a fixed connection between the substrate and the card holder body.

[0026] Optionally, the substrate is provided with a plurality of sensor mounting holes located within the sealing groove;

[0027] Both the substrate and the cover plate include light-transmitting materials.

[0028] Optionally, the cover plate is provided with a plurality of air inlets, which are disposed through the cover plate;

[0029] The air inlet is connected to the culture chamber, and a one-way gas valve is installed inside the air inlet.

[0030] Optionally, a needle-shaped air tube is provided inside the air inlet, with one end of the needle-shaped air tube connected to the air inlet and the other end located inside the cell filter.

[0031] Beneficial effects:

[0032] This application provides a microfluidic cartridge for automated cell perfusion culture. The cartridge comprises a substrate, a cartridge body, and a cover. The cartridge body includes at least one culture chamber with a cell filter. The culture chamber includes a perfusion inlet and a perfusion outlet. The cartridge body includes a perfusion fluid inlet and a perfusion fluid outlet. The perfusion fluid inlet and the perfusion outlet are connected via perfusion conduits, and the perfusion outlet and the perfusion fluid outlet are connected via an outlet conduit. In a first direction, the perfusion outlet is closer to the substrate than the perfusion inlet, and the outlet conduit is closer to the substrate than the perfusion conduit. When using this microfluidic cartridge, a cell sample is placed within the cell filter of the culture chamber. Culture medium is injected into the culture chamber via the perfusion fluid inlet, the perfusion conduit, and the perfusion inlet. Cell metabolic products are discharged via the perfusion outlet, the outlet conduit, and the perfusion fluid outlet. The positional differences between the perfusion inlet and the perfusion outlet, as well as between the perfusion conduit and the outlet conduit, prevent backflow of the culture medium. Combined with a one-way filter membrane, the flow direction of the culture medium during perfusion culture and batch feeding is automatically controlled, avoiding crosstalk and cross-contamination.

[0033] The culture chambers are small (less than 5 mL) and equipped with cell filters to separate cells from newly added culture medium and provide an attachment substrate for cell clusters. The perfusion medium enters from a higher position to provide nutrients, while target metabolites or old culture medium flow out from a lower position; this enables dynamic cell culture. The culture chambers are completely transparent from top to bottom and, through integrated sensor foils, can monitor state parameters such as pH, OD, oxygen, and carbon dioxide in the culture medium online, maintaining functionality consistent with traditional large-scale fermenters. Furthermore, this microfluidic cartridge can provide cell perfusion culture capabilities, enabling the culture of cell lines across multiple growth cycles and realistically simulating the continuous blood supply environment within cells, reconstructing the physiological environment of cell lines or cell populations in vivo. This can increase cell culture throughput, shorten fermentation time, and reduce the volume of the culture system. Automated perfusion culture across multiple culture chambers not only allows for precise control of culture conditions but also increases the throughput requirements of related quantitative experiments. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1A This is a schematic diagram of the overall structure of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application;

[0036] Figure 1B This is a bottom view of the structure of a microfluidic cartridge for automated cell perfusion culture hidden on a substrate, according to an embodiment of this application.

[0037] Figure 2 This is a schematic diagram of the main body of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application;

[0038] Figure 3 yes Figure 1A A sectional view of section EE;

[0039] Figure 4 yes Figure 1A A sectional view of section FF in the middle;

[0040] Figure 5 This is a schematic diagram of the structure of a cell filter in a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application;

[0041] Figure 6This is a perspective view of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application;

[0042] Figure 7 A is a bottom view of the substrate of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application;

[0043] Figure 7 B is a top view of the substrate of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application.

[0044] Figure 8 yes Figure 1B Sectional view of section DD;

[0045] Figure 9 A is a bottom view of the cover plate of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application.

[0046] Figure 9 B is a top view of the cover plate of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application.

[0047] Figure 10 This is a cross-sectional schematic diagram of a liquid check valve assembly of a microfluidic cartridge for automated cell perfusion culture according to an embodiment of this application.

[0048] Figure 11 This is a schematic diagram of the structure of an environmental detection and control device according to an embodiment of this application.

[0049] Explanation of reference numerals in the attached drawings: 1. Substrate; 11. Sealing ring; 111. Sealing groove; 112. Screw hole; 113. Countersunk hole; 151. First sensor mounting hole; 152. Second sensor mounting hole; 153. Second sensor mounting hole; 2. Cartridge body; 21. Culture chamber; 22. Perfusion fluid inlet; 23. Perfusion fluid outlet; 222. Perfusion inlet; 232. Perfusion outlet; 223. Perfusion pipe; 233. Outlet pipe; 224. Perfusion downward bend pipe; 234. Perfusion upward bend pipe; 26. Slot; 28. Magnet tube; 3. Cover plate; 31. Air inlet; 32. Gas one-way valve; 34. Double-sided tape; 35. Needle-shaped gas tube; 4. Liquid one-way valve assembly; 41. External one-way valve assembly; 42. Nut; 43. Screw; 44. 45. Inner O-ring seal; 46. Outer O-ring seal; 47. One-way filter membrane; 48. Inner one-way valve assembly; 59. Liquid line connector; 50. Outlet connector; 51. Dissolved oxygen sensor; 52. pH sensor; 53. Carbon dioxide sensor; 60. Cell filter; 61. Opening; 62. Protrusion; 63. Filter screen; 64. Micropore; 70. Mechanical control platform; 71. Electric stage; 72. Temperature sensor; 73. Transparent temperature control plate; 8. Controller; 91. Environmental detection and control device; 92. Light source; 92. First sensor detector; 92. Second sensor detector; 92. Third sensor detector; 90. First color filter group; 90. Second color filter group; 90. Third color filter group; 93. Focusing lens. Detailed Implementation

[0050] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0051] In related technologies, the culture and screening of cell lines and cell clusters mainly rely on microplates, petri dishes, or shake flasks. Microplates and petri dishes result in long cell growth cycles and low biomass yields, while shake flasks can shorten culture or fermentation time; however, none of these methods can monitor parameters during the culture process in real time. Furthermore, existing cell culture methods based on microplates and shake flasks require manual or periodic feeding via pipettes, thus primarily employing batch culture / fermentation or fed-batch culture / fermentation models, which cannot simulate the continuous perfusion culture or fermentation modes of large and medium-sized fermenters. Large fermenters can provide batch production modes such as continuous perfusion culture and fed-batch fermentation, but they require a large volume of experimental samples.

[0052] In view of this, this application proposes a microfluidic cartridge for automated cell perfusion culture. The cartridge comprises a substrate, a cartridge body, and a cover. The cartridge body includes at least one culture chamber containing a cell filter. The culture chamber includes a perfusion inlet and a perfusion outlet. The cartridge body includes a perfusion fluid inlet and a perfusion fluid outlet. The perfusion fluid inlet and the perfusion outlet are connected via a perfusion conduit, and the perfusion outlet and the perfusion fluid outlet are connected via an outlet conduit. In a first direction, the perfusion outlet is closer to the substrate than the perfusion inlet, and the outlet conduit is closer to the substrate than the perfusion conduit. When using this microfluidic cartridge, a cell sample is placed inside the cell filter of the culture chamber, and perfusion is performed... The inlet, perfusion conduit, and perfusion outlet allow for the injection of culture medium into the culture chamber, while the outlet, outlet conduit, and perfusion outlet allow for the removal of cell metabolic products. The positional differences between the perfusion inlet and outlet, as well as between the perfusion conduit and outlet conduit, prevent backflow of the culture medium, thus achieving unidirectional flow of the culture medium. In this way, dynamic cell culture is achieved as a whole. Furthermore, this microfluidic cartridge can be used to complete cell line culture for multiple growth cycles and realistically simulate the continuous blood supply environment within cells, reconstructing the physiological environment of cell lines or cell populations in vivo. This can increase cell culture throughput, shorten fermentation time, and reduce the volume of the culture system.

[0053] Reference Figure 1A As shown in the figure, a microfluidic cartridge for automated cell perfusion culture disclosed in this application embodiment includes a substrate 1, a cartridge body 2, and a cover plate 3.

[0054] Specifically, refer to Figure 1A and Figure 2 As shown, the card holder body 2 is disposed on the substrate 1, and the cover plate 3 is disposed on the side of the card holder body 2 opposite to the substrate 1. The card holder body 2 is generally rectangular in shape and includes at least one culture chamber 21. The culture chamber 21 penetrates the card holder body 2 in a first direction. It can be understood that the culture chamber 21 is a hole in the card holder body 2. After its two ends are closed by the substrate 1 and the cover plate 3, a closed culture chamber 212 is formed. The cross-sectional shape of the culture chamber 21 can be circular or square, etc. In the embodiment of this application, the cross-sectional shape of the culture chamber 21 is square. The first direction is the direction perpendicular to the substrate 1.

[0055] In practical applications, substrate 1 can be manufactured by laser cutting or machining. The material of substrate 1 can include PMMA, PC, or COP, giving substrate 1 good light transmittance. Cover plate 3 can be manufactured by laser cutting or machining. The material of cover plate 3 can include silicon, glass, quartz, or plastic, giving cover plate 3 good light transmittance. Thus, the upper and lower surfaces of the penetration surface of culture chamber 21 are transparent materials, and there are no other obstructions or interferences except for cell filter 6, which facilitates imaging and detection.

[0056] Reference Figure 2 As shown, the side wall of the culture chamber 21 includes a perfusion inlet 222 and a perfusion outlet 232. At the same time, the four side walls of the cartridge body 2 are provided with perfusion fluid inlets 22 and perfusion fluid outlets 23. The perfusion fluid inlets 22 and 222 are connected by perfusion pipes 223, and the perfusion fluid outlets 23 and 232 are connected by outlet pipes 233. In this way, the culture medium can enter the perfusion channel 223 through the perfusion fluid inlet 222, and then enter the culture chamber 21 through the perfusion inlet 222. The cell growth metabolites will enter the outlet pipe 233 through the perfusion outlet 232, and finally be discharged through the perfusion fluid outlet 23.

[0057] It should be noted that the cartridge body 2 can be manufactured using additive manufacturing processes, such as 3D printing. Therefore, the culture chamber 21, perfusion inlet 222, perfusion outlet 232, perfusion fluid inlet 22, perfusion fluid outlet 23, perfusion pipe 223, and outlet pipe 233 can all be formed during the printing process. The specific preparation process will not be elaborated in detail in this embodiment. The material of the cartridge body 2 may include PLA or ABS, and the cartridge body 2 may be made of transparent, translucent, or opaque materials.

[0058] Meanwhile, in the first direction, the perfusion outlet 232 is closer to the substrate 1 than the perfusion inlet 222, and the outlet pipe 233 is closer to the substrate 1 than the perfusion pipe 223. This creates a certain liquid level difference between the perfusion outlet 232 and the perfusion inlet 222, and between the outlet pipe 233 and the perfusion pipe 223. This can prevent backflow of the culture medium liquid and ensure unidirectional flow of the culture medium liquid.

[0059] Furthermore, referring to Figure 3 and Figure 4As shown, a downward-curved irrigation pipe 224 is provided between the irrigation inlet 222 and the irrigation pipe 223. One end of the downward-curved irrigation pipe 224 is connected to the irrigation inlet 22, and the other end is connected to the irrigation pipe 223. An upward-curved irrigation pipe 234 is provided between the irrigation outlet 232 and the outlet pipe 233. One end of the upward-curved irrigation pipe 234 is connected to the irrigation outlet 232, and the other end is connected to the outlet pipe 233. The downward-curved irrigation pipe 224 and the upward-curved irrigation pipe 234 can better ensure the unidirectional flow of the culture medium liquid.

[0060] Furthermore, referring to Figure 4 As shown, the microfluidic cartridge also includes multiple liquid inlet connectors 5 and multiple outlet connectors 50. The liquid inlet connectors 5 are connected to the perfusion fluid inlet 22, and the outlet connectors 50 are connected to the perfusion fluid outlet 23. Furthermore, the liquid inlet connectors 5 and outlet connectors 50 are connected to an external multi-channel perfusion fluid control device. Using the multi-channel perfusion fluid control device, automated injection and outflow of culture medium liquid can be achieved, making the use of the microfluidic cartridge more convenient. The material of the liquid inlet connectors 5 may include polytetrafluoroethylene (PTFE) or PTFE.

[0061] Furthermore, referring to Figure 1B and Figure 5 As shown, a cell filter 6 is provided in the culture chamber 21. The cell filter 6 is configured as a cup-shaped structure with one end open, and at least one filter 63 is provided on the side wall or the bottom wall away from the opening 61 of the cup-shaped structure.

[0062] Specifically, the filter 63 can be honeycomb-shaped, comprising multiple micropores 631, with the pore size of the micropores 631 being smaller than the diameter of the target cultured cells within the cell filter 6. This separates the cells from the outer culture medium while maintaining good solution permeability. Newly added culture medium enters through the higher perfusion inlet 222 and passes through the filter 63, providing nutrients to the cell line or cell cluster. Target metabolites or old culture medium flow out through the lower perfusion outlet 232 via the filter 63. Simultaneously, the cell filter 6 can also provide an attachment matrix for cell cluster culture. Cells and matrix gel for cell cluster culture can be pre-placed at the bottom of the cell filter 6. Culture medium solution is immersed into the cells from the surrounding and / or bottom of the filter 63, providing the necessary nutrients for cell growth and enabling three-dimensional culture.

[0063] In practical applications, filter 63 can utilize polycarbonate membranes with different pore sizes and treatments, ranging from 0.4 micrometers to 3 micrometers. Treatment methods for the polycarbonate membrane include, but are not limited to, hydrophilic treatment and fibronectin coating. Furthermore, in this embodiment, cell filter 6 is a disposable sterile consumable that can be sterilized in batches after assembly for later use, or commercially available consumables can be used. The material of cell filter 6 can include biocompatible plastics, aluminum alloys, or polytetrafluoroethylene, giving cell filter 6 certain high-temperature resistance and acid / alkali resistance properties. In this application, cell filter 6 is a disposable sterile consumable that can be sterilized in batches after manufacturing and assembly for later use.

[0064] Furthermore, referring to Figure 2 and Figure 5 As shown, a protrusion 62 is provided on the upper part of the cell filter 6, and a slot 26 adapted to the protrusion is provided on the upper part of the culture chamber 21. By embedding the protrusion 62 on the cell filter 6 into the slot 26, the cell filter 6 can be fixed in the culture chamber 21.

[0065] The microfluidic cartridge provided in this application embodiment enables dynamic cell culture. Furthermore, the microfluidic cartridge can be used to complete cell line culture for multiple growth cycles and realistically simulate the continuous blood supply environment within cells, reconstructing the physiological environment of cell lines or cell populations in vivo. This can increase cell culture throughput, shorten fermentation time, and reduce the volume of the culture system.

[0066] Furthermore, this microfluidic cartridge can be sterilized, aseptically handled in a laminar flow fume hood, and used for subsequent aseptic culture, allowing for sample collection and further analysis. It also has the potential for hardware and software integration with laboratory robots or other equipment.

[0067] In one alternative implementation, refer to Figure 6 As shown, multiple culture chambers 21 are provided, and the multiple culture chambers 21 are distributed in an array at certain intervals within the cartridge body 2.

[0068] Specifically, in this embodiment, multiple perfusion inlets 22 and perfusion pipes 223 are provided, with each perfusion inlet 222 corresponding one-to-one with a perfusion inlet 222 of a culture chamber 21, and each perfusion pipe 223 corresponding one-to-one with a perfusion inlet 222 of a culture chamber 21. That is, each culture chamber 21 is injected with culture medium liquid through a separate perfusion inlet 22 and perfusion pipe 223, allowing different culture medium liquids to be independently injected into different culture chambers 21 for parallel testing under different experimental conditions. Multiple perfusion inlets 22 and multiple perfusion outlets 23 are respectively located on the four sides of the cartridge body 2.

[0069] Meanwhile, multiple perfusion outlets 23 and outlet pipes 233 are provided, with each perfusion outlet 23 corresponding to a specific outlet pipe 233. The multiple outlet pipes 233 and multiple perfusion pipes 223 can be horizontally staggered within the cartridge body 2, including perfusion pipes 223 located at higher positions and outlet pipes 233 located at lower positions. The perfusion outlets 232 of multiple culture chambers 221 located in the same column or row are connected to one of the outlet pipes 233. In this way, culture medium waste liquid generated by cells in the culture chambers 21 located in the same column or row can be discharged all at once through the same outlet pipe 233, thereby improving the discharge efficiency of the culture medium waste liquid.

[0070] In this embodiment of the application, there are 4*5 culture chambers 21 and 4 outlet pipes 233. Therefore, each outlet pipe 233 is connected to the perfusion outlet 23 of the 5 culture chambers 21.

[0071] In one alternative implementation, refer to Figure 7 As shown in Figure B, at least one closed sealing groove 111 is provided on the substrate 1, the culture chamber 21 is located in the corresponding sealing groove 111, and a sealing ring 11 is provided in the sealing groove 111.

[0072] Specifically, the shape of the sealing groove 111 may include a circle or a square, etc. In this embodiment, the sealing groove 111 is circular. After the cartridge body 2 is connected to the substrate 1, each culture chamber 21 is located in the closed area enclosed by the closed sealing groove 111 and is sealed by the corresponding sealing ring 11; thereby increasing the overall sealing between the substrate 1 and the culture chamber 21.

[0073] Furthermore, referring to Figure 7 B and Figure 8 As shown, at least one set of fasteners is provided on the substrate 1, each set of fasteners corresponding to each sealing groove 111. The fasteners include multiple screws 43, which pass through screw holes 112 on the substrate 1, and the multiple screws 43 are located on the outside of the sealing groove. At the same time, a magnetic tube 28 is provided around each culture chamber on the cartridge body 2, corresponding to the screws in the set of fasteners and embedded therein. After the screws 43 pass through the magnetic tubes 28, the substrate 1 and the cartridge body 2 can be fixedly connected.

[0074] Preferably, each set of fasteners described in this application may include four screws 43 and four magnet tubes 28, which are respectively disposed at the four corners of the corresponding sealing groove 111.

[0075] In this way, multiple sealing grooves 111 are arranged opposite to the culture chamber 21, each sealing groove 111 is fixed with a sealing ring 11, and the corresponding fasteners cooperate one by one to seal the surrounding environment area at the bottom of the culture chamber 21 under the pre-tightening force of magnetic adsorption; and to achieve overall liquid sealing between the microfluidic cartridge body 2 and the microfluidic cartridge substrate, such as Figure 2 As shown. The multiple screws 43 and magnetic tubes 48 provided in this application can complete the fixing and sealing of all holes at once, avoiding the need to manually tighten a large number of screws during subsequent use; the fixing is reversible, and by applying appropriate external force to the card holder body 2 and the base plate 1, the magnetic fixation can be separated for quick disassembly.

[0076] In practical applications, the screw hole 112 can be an M2 hexagon socket screw hole, and the corresponding screw 43 can be an M2 hexagon socket screw. The sealing groove 111 can be set as a sealing groove with an inner diameter of 14.5 mm and a line width of 1.2 mm. The magnetic tube 28 is a hollow annular round tube, and the screw 43 can be inserted into the central hole and magnetically attracted, providing mutual attraction under the action of magnetic attraction. The magnetic tube 28 can be embedded into the microfluidic card cartridge body 2 during the additive manufacturing process.

[0077] At the same time, refer to Figure 7 As shown in Figure A, on the side of the substrate 1 away from the card holder body 2, a plurality of countersunk holes 113 are provided opposite to the screw holes. The shape of the countersunk holes 113 may be cylindrical or waist-shaped. The countersunk holes 113 can be used to pre-fix screws 43 and make the top of the screws 43 sink into the hole without protruding from the surface, so as to ensure a smooth bottom surface of the substrate 1 after assembly.

[0078] Furthermore, each sealing groove 111 on the substrate 1 is provided with multiple sensor fixing holes.

[0079] Specifically, refer to Figure 7 As shown in Figure B, the multiple sensor mounting holes may include a mounting hole 151 for fixing a first sensor, a mounting hole 152 for fixing a second sensor, and a mounting hole 153 for fixing a third sensor. The sensor mounting holes 152, 152, and 153 are concave holes with dimensions corresponding to the corresponding sensors.

[0080] For example, refer to Figure 3 As shown in the embodiment of this application, the sensor fixing hole 151 is used to attach and fix the dissolved oxygen sensor 51, the sensor fixing hole 152 is used to attach and fix the pH sensor 52, and the sensor fixing hole 152 is used to attach and fix the carbon dioxide sensor 53. The dissolved oxygen sensor 51, pH sensor 52 and carbon dioxide sensor 53 are all thin film foils used to dynamically detect the environmental conditions of cell culture.

[0081] In one alternative implementation, refer to Figure 9As shown in Figure B, a plurality of air inlets 31 are provided on the cover plate 3 opposite to the culture chamber 21. The air inlets 31 penetrate the cover plate 3 and are connected to the culture chamber 21. A one-way gas valve 32 is provided on the outside of the air inlets 31.

[0082] Specifically, each air inlet 31 corresponds one-to-one with a culture chamber 21. A one-way gas valve 32 allows gas to flow unidirectionally towards the air inlet 31 to avoid mutual interference. Furthermore, the one-way gas valve 32 can be independently connected to an external multi-channel gas path control device. For example, the one-way gas valve 32 can be a membrane one-way valve with a sterile filter. During cell culture, the multi-channel gas path control device can control the amount and proportion of ambient gas flowing to the one-way gas valve 32, providing the necessary gaseous environment for cell culture; the ambient gas may include a combination of gases such as carbon dioxide, oxygen, and nitrogen.

[0083] Furthermore, referring to Figure 9 As shown in Figure A, a needle-shaped trachea 35 is provided inside the air inlet 31. One end of the needle-shaped trachea 35 is connected to the air inlet 31, and the other end is located inside the cell filter 6.

[0084] Specifically, during cell perfusion culture, the needle-shaped trachea 35 can introduce the ambient gas introduced by the gas one-way valve 32 into the solution inside the cell filter 6, thereby promoting cell growth.

[0085] Furthermore, referring to Figure 9 As shown in Figure A, the back of the cover plate 3 is provided with pre-applied double-sided adhesive 34, and the area of ​​double-sided adhesive 34 can cover the area outside the multiple culture chambers 21 of the card holder body 2. The double-sided adhesive 34 enables reversible sealing between the cover plate 3 and the card holder body 2. The double-sided adhesive 34 can be a high-temperature resistant, medium-viscosity, and medium-peel-strength double-sided adhesive.

[0086] In one alternative implementation, refer to Figure 4 As shown, the microfluidic cartridge also includes a liquid check valve assembly 4, which is connected to the irrigation fluid inlet 22 and is located between the irrigation fluid inlet 22 and the liquid connector 5.

[0087] Specifically, refer to Figure 10 As shown, the liquid check valve assembly 4 may include an outer check valve assembly 41, a nut 42, an internal hex screw 43, an inner O-ring seal 44, an outer O-ring seal 45, a one-way filter membrane 46, and an inner check valve assembly body 47. The nut 42 and the internal hex screw 43 fix the outer check valve assembly 41 and the inner check valve assembly 47 together to form the housing of the entire liquid check valve assembly 4.

[0088] The outer one-way valve assembly 41 has an inner O-ring groove for placing the inner O-ring to prevent waste liquid from leaking out between the outer one-way valve assembly 41 and the inner one-way valve assembly 47, and its front end has an M2 thread; the front end of the outer one-way valve assembly 41 has an M2 stud for fitting the outer O-ring 45 into the stud, and the stud is fixedly connected to the microfluidic cartridge body 2 to achieve end face sealing and prevent waste liquid from leaking out from the connection. The inner one-way valve assembly 47 has a threaded hole inside for connecting to the liquid line connector 5 for the connection between the two.

[0089] The external one-way valve assembly 41 has a filter tank for placing the one-way filter membrane 46. The one-way filter membrane 46 is placed in the filter tank and has different flow resistances on both sides. Specifically, the side of the one-way filter membrane 46 facing the perfusion fluid inlet 22 is the first side, and the side of the one-way filter membrane 46 away from the perfusion fluid inlet 22 is the second side. When liquid permeates the one-way filter membrane 46, the flow resistance of liquid permeating from the second side to the first side is less than the flow resistance of liquid permeating from the first side to the second side. Therefore, the one-way filter membrane 46 can intercept useful free nutrients in the culture medium waste liquid within the culture chamber 21, thereby limiting the flow direction of the liquid and avoiding cross-contamination.

[0090] In an optional implementation, a multi-parameter online monitoring sensor may also be provided, including a dissolved oxygen sensor 51, a pH sensor 52, and a carbon dioxide sensor 53, for detecting the environmental conditions of the cultured cells.

[0091] Specifically, within the end face of the sealing ring 11 at the bottom of the pore in the culture chamber 21, luminescent dyes sensitive to pH, oxygen, and carbon dioxide are pre-embedded as dissolved oxygen sensor 51, pH sensor 52, and carbon dioxide sensor 53, respectively. They are then excited by a bottom light source, and the emitted fluorescence decay signal (based on the fluorescence quenching effect) is measured simultaneously. The actual value of the physical quantity to be measured is obtained by solving the control group and the linear group.

[0092] The principle of the sensor described in this application embodiment is based on the principle of fluorescence quenching. For example, when blue light shines on a fluorescent material, it excites the material and emits red light. Since sensitive molecules (e.g., oxygen molecules) can carry away energy, the duration and intensity of the excited red light are inversely proportional to the concentration of the sensitive molecules. By measuring the phase difference between the excited red light and the reference light and comparing it with the internal calibration value, the concentration of the sensitive molecules can be calculated.

[0093] For example, the dissolved oxygen sensor 51 used in this application is an Oxygen Sensor Foil SF-RPSu4, the pH sensor 52 is a pH Sensor Foils, and the carbon dioxide sensor 53 is a CO2 Sensor Foil SF-CD1R. These sensors are all based on the principle of fluorescence quenching. A special polyester foil of platinum metalloporphyrin complex (which allows gas molecules to pass through) is cut to the size of the sensor fixing holes 151, 152, and 153, and fixed with the probe side facing down using transparent adhesive. The transparent adhesive can be SG2Silicone Glue, transparent, or ColorCode.

[0094] In this embodiment, the culture chamber 21 is a through hole, with transparent material on both the top and bottom of the through surface. A bottom transparent window is reserved for the detection of the aforementioned sensor. Fluorescence excitation and imaging methods can be used to scan and measure parameters such as OD value, dissolved oxygen (DO), pH value, and carbon dioxide online, and the temperature of the microfluidic cartridge can be controlled. This can improve the quantitative experiments related to cell line and cell cluster culture and increase the throughput of perfusion experiments.

[0095] Based on the above, the microfluidic cartridge for automated cell perfusion culture described in this application is used as follows:

[0096] 1. First, install the liquid check valve assembly 4 externally.

[0097] 2. Then, three sensor foils (dissolved oxygen sensor 51, pH sensor 52 and carbon dioxide sensor 53) are attached to the fixing holes 151 / 152 / 153 at 120° intervals inside the sealing groove 111 on the substrate 1 to detect the environmental conditions of the cultured cells. The substrate 1 is then connected and fixed to the microfluidic cartridge body 2 with sealing ring 11 and screw 43.

[0098] 3. Next, assemble and connect the liquid line connector 5 and the liquid one-way valve assembly 4, and connect the two assemblies to the corresponding infusion liquid inlet 22 in the cartridge body 2. Connect the remaining liquid line connectors 5 to the corresponding infusion liquid outlet 23 in the cartridge body 2 to complete the assembly.

[0099] 4. Then place the cover plate 3 with the needle-like endotracheal tube 35 installed on the upper part of the card box body 2, and sterilize the whole thing.

[0100] 5. Next, cell seeding is performed in a sterile operating room, including two scenarios:

[0101] 5A. For cell cluster culture such as organ-on-a-chip, a certain number of cells to be cultured are obtained from animal cells or tissues through separation, extraction, purification and other steps, and mixed with culture medium, such as matrix gel (hydrogel), in a certain proportion, and added together to a pre-sterilized cell filter 6.

[0102] 5B. For cell line culture, such as yeast and mammalian cells, add the revived or pre-cultured cell line to a pre-sterilized cell filter 6.

[0103] 6. Next, in the sterile operating table, place the cell filter 6 into the protrusion 62 of the cartridge body 2, and peel off the double-sided tape used for fixing the cover. Then connect the cover and the cartridge body 2 with double-sided tape, and install the pre-sterilized gas one-way valve 32 into the corresponding air inlet 31 of the cover.

[0104] 7. Finally, insert the perfusion and waste liquid pipes of the multi-channel perfusion fluid control device into the liquid connector 5 and the outlet connector 50 respectively, and connect the gas pipes of the external multi-channel gas control device to the gas check valve 32 respectively; set the corresponding parameters, and deliver the culture medium, carbon dioxide gas, etc. to the culture chamber 21 of the cartridge body 2 for cell culture.

[0105] Based on the above, after the microfluidic cartridge used for automated cell perfusion culture is used, the substrate 1, cartridge body 2, partial liquid check valve assembly 4, and liquid circuit connector 5 are expected to undergo the following regeneration steps before reuse:

[0106] 1. Acid and alkali soaking increases the solubility of microorganisms and culture medium residues adhering to the micropores;

[0107] 2. Ultrasonic cleaning removes microorganisms and culture medium residues adhering to the micropores.

[0108] 3. Multiple water rinses and centrifugal drying;

[0109] 4. The substrate 1 and the card holder body 2 are sterilized separately. The sterilization scheme can be as follows:

[0110] 4a) Sterilization by pressure steam;

[0111] 4b) Sterilization methods such as ethylene oxide and plasma radiation are used.

[0112] 5. Assemble the components in a sterile environment and seal the gas inlet with a 0.22µm membrane.

[0113] Reference Figure 11As shown in the embodiments of this application, a culture environment detection and control device is also disclosed. The culture environment detection and control device 9 includes a mechanical control platform 7, an illumination source 91, a first sensor detector 921, a second sensor detector 922, a third sensor detector 923, a first color filter group 901, a second color filter group 902, a third color filter group 903, a focusing lens 93, etc.

[0114] The first sensor detector 921 is used in conjunction with the first color filter group 901 and the dissolved oxygen sensor 51. The first sensor detector 921 can emit dissolved oxygen detection excitation light, which illuminates the dissolved oxygen sensor 51 at the bottom of the hole in the culture chamber 21, and the first color filter group 901 selects the corresponding fluorescence to the surface detector of the first sensor detector 921.

[0115] The second sensor 922 works in conjunction with the second filter group 902 and the pH sensor 52. The second sensor 922 can emit dissolved oxygen detection excitation light, which illuminates the pH sensor 52 at the bottom of the well in the culture chamber 21, and the first filter group 902 selects the corresponding fluorescence to the surface detector of the second sensor 922.

[0116] The third sensor detector 923 works in conjunction with the third color filter group 903 and the carbon dioxide sensor 53. The third sensor detector 921 can emit carbon dioxide detection excitation light, which illuminates the third carbon dioxide sensor 53 at the bottom of the well in the culture chamber 21, and the third color filter group 903 selects the corresponding fluorescence to the surface detector of the third sensor detector 923.

[0117] In this application, the illumination source 91 provides illumination light for cell absorbance detection. Illumination light with a wavelength of 600nm can be selected. The light is irradiated into the culture chamber 21 through the focusing lens 93, and the transmitted light is detected by the second sensor detector 922 to obtain the cell culture medium concentration information (OD600) of the culture chamber. At this time, the excitation light of the second sensor detector 922 is turned off, and only detection is performed.

[0118] The culture environment detection and control device 9 of this application uses a controller 8 to acquire and control signals. In the signal acquisition, a red light source synchronized with the excitation light can be used as a reference to measure the phase difference between the excitation light and the reference light and compare it with the internal calibration value to calculate the concentration of the sensitive molecule. After linearization and temperature compensation, the final value is output.

[0119] Reference Figure 11As shown, the mechanical control platform 7 of this application is an electrically driven stage with temperature control function, including an electrically driven stage 73, a transparent temperature control plate 75, and a temperature sensor 72, used for temperature control, movement, and positioning of the microfluidic cartridge, as well as oscillation and mixing. The transparent temperature control plate 75 is located below the microfluidic cartridge and can be heated, preferably using ITO transparent glass; the temperature sensor 72 can be set inside the culture chamber 21. The electrically driven stage 73 carries the microfluidic cartridge and moves precisely along the horizontal XY axis, and assists the culture environment detection and control device 9 in detecting different culture chambers 21. Continuous oscillation occurs throughout the entire culture process, making it suitable for both aerobic and anaerobic cultures.

[0120] In this application, the temperature during cell culture can be controlled in a closed loop using a transparent temperature control plate 75, a temperature sensor 72, and a controller 8. At the same time, each well can be independently vented with ambient gas, which can be a combination of carbon dioxide, oxygen, and nitrogen. Therefore, in conjunction with pH, ​​dissolved oxygen, and carbon dioxide sensors pre-embedded in the solution, closed-loop control of parameters such as dissolved oxygen can also be achieved.

[0121] With the aforementioned culture environment detection and control device, each culture chamber is small in volume (less than 5 ml), enabling automated detection and control operations during perfusion culture with little or no human supervision, saving labor costs during experiments. Through continuous sample injection via microfluidic pipelines and pneumatic injection, batch feeding and continuous feeding functions are realized. pH, oxygen, and carbon dioxide sensors are integrated at the bottom of the culture chamber to monitor biological process variables at high resolution, and it has scalability to standard fermenters and laboratory-scale bioreactors.

[0122] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0123] It should also be noted that, in this document, the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and 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, and therefore should not be construed as a limitation of this application. Furthermore, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations, nor should they be construed as indicating or implying relative importance. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device 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 terminal device. In the absence of further restrictions, an element defined by the phrase "includes a..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes the element.

[0124] The technical solutions provided in this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand this application, and the content of this specification should not be construed as a limitation of this application. Furthermore, for those skilled in the art, there will be different forms of changes in the specific implementation methods and application scope based on this application. It is neither necessary nor possible to exhaustively list all implementation methods here, and obvious changes or modifications derived therefrom are still within the protection scope of this application.

Claims

1. A microfluidic cartridge for automated cell perfusion culture, characterized in that, include: Substrate, card holder body and cover; The card holder body is disposed on the substrate, and the cover plate is disposed on the side of the card holder body opposite to the substrate; The cartridge body includes at least one culture chamber, the culture chamber extends through the cartridge body in a first direction, and a cell filter is disposed inside the culture chamber; The culture chamber includes an inlet and an outlet, and the inlet and outlet are connected to the culture chamber. The main body of the card box also includes a side-mounted irrigation fluid inlet and an irrigation fluid outlet, an internal irrigation pipe and an outlet pipe, the irrigation inlet and the irrigation fluid inlet are connected by the irrigation pipe, and the irrigation outlet and the irrigation fluid outlet are connected by the outlet pipe; In the first direction, the irrigation outlet is closer to the substrate than the irrigation inlet, and the outlet pipe is closer to the substrate than the irrigation pipe; the first direction is perpendicular to the substrate. An irrigation downbend pipe is provided between the irrigation inlet and the irrigation pipe, with one end of the irrigation downbend pipe connected to the irrigation inlet and the other end connected to the irrigation pipe; An irrigation upbend pipe is provided between the irrigation outlet and the outlet pipe, with one end of the irrigation upbend pipe connected to the irrigation outlet and the other end connected to the outlet pipe; The culture chambers are provided in multiple ways, and the multiple culture chambers are arrayed on the cartridge body; The perfusion fluid inlet is provided in multiple ways, and each of the multiple perfusion fluid inlets corresponds one-to-one with the perfusion inlet of the multiple culture chambers; the perfusion pipe is provided in multiple ways, and each of the multiple perfusion pipes corresponds one-to-one with the perfusion inlet of the multiple culture chambers. The perfusion fluid outlet and the outlet pipe are provided in multiple ways, and the multiple perfusion fluid outlets correspond one-to-one with the multiple outlet pipes; the perfusion outlets of the multiple culture chambers located in the same column or the same row are connected to one of the multiple outlet pipes. The cell filter is configured as a cup-shaped structure with one end open, and at least one filter is provided on the side wall or the bottom wall away from the opening of the cup-shaped structure. The pore size of the filter is smaller than the diameter of the target cultured cells inside the cell filter.

2. The microfluidic cartridge for automated cell perfusion culture according to claim 1, characterized in that, The microfluidic cartridge also includes: A liquid check valve assembly, wherein the liquid check valve assembly is connected to the perfusion fluid inlet; The liquid one-way valve assembly includes a one-way filter membrane, the side of the one-way filter membrane facing the perfusion fluid inlet is the first side, and the side of the one-way filter membrane away from the perfusion fluid inlet is the second side. Specifically, when liquid permeates the unidirectional filter membrane, the flow resistance from the second side to the first side is less than the flow resistance from the first side to the second side.

3. The microfluidic cartridge for automated cell perfusion culture according to any one of claims 1-2, characterized in that: The substrate is provided with at least one closed sealing groove, the culture chamber is located in the corresponding sealing groove, and a sealing ring is provided in the sealing groove.

4. The microfluidic cartridge for automated cell perfusion culture according to claim 3, characterized in that: At least one set of fasteners is provided on the substrate, the fasteners including a plurality of screws, the screws passing through the substrate, and the plurality of screws being located outside the sealing groove; The card holder body is embedded with a magnetic tube that corresponds one-to-one with the screws. The screws can be inserted into the center of the magnetic tube to achieve a fixed connection between the substrate and the card holder body.

5. The microfluidic cartridge for automated cell perfusion culture according to claim 3, characterized in that: The substrate has multiple sensor mounting holes located within the sealing groove; Both the substrate and the cover plate include light-transmitting material.

6. The microfluidic cartridge for automated cell perfusion culture according to any one of claims 1-2, characterized in that: The cover plate is provided with multiple air inlets, which are disposed through the cover plate; The air inlet is connected to the culture chamber, and a one-way gas valve is provided outside the air inlet.

7. The microfluidic cartridge for automated cell perfusion culture according to claim 6, characterized in that: A needle-shaped air tube is provided inside the air inlet, with one end of the needle-shaped air tube connected to the air inlet and the other end located inside the cell filter.

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