Organoid culture chip

Abstract

The present invention discloses an organoid culture chip, which comprises a pressing plate, an upper glass sheet, a flow channel layer sheet, a lower glass sheet, a biosensor and a packaging bottom plate arranged in sequence from top to bottom; the flow channel layer sheet includes a sheet body and two culture units arranged side by side on the sheet body; the biosensor is in the shape of a thin sheet, and its end has a detection sheet for monitoring biological parameters of the organoids in the culture holes in the culture unit and providing reverse electrical stimulation. The organoid culture chip provided by the present invention can realize real-time detection of various biological parameter indexes of organoids during the culture process by arranging biosensors in some of the culture holes, and can provide reverse electrical stimulation to regulate the developmental function of organoids. Moreover, the biosensor of the present invention adopts a flat side wire-out structure, which is convenient for wiring and does not affect the sealing of the culture chamber; by arranging an upper and lower light-transmitting structure in some other culture holes, optical observation and Raman spectroscopy measurement can be carried out in these culture holes.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and particularly to an organoid culture chip. Background Art

[0002] Organoids are formed by three-dimensional (3D) culture of cells with stem cell potential, thus forming partial tissues of corresponding organs. Organoids can accurately simulate in vivo epithelial structures and can be passaged and cultured for a long time. The purpose of these models is to explain the functions of the body from the molecular level to cells, tissues, organs, or the whole organism. Currently, organoid models of human and mouse stomach, small intestine, liver, pancreas, prostate, etc. have been successfully established. These organoid models can all be cultured for a long time and have stable phenotypic and genetic characteristics. Organoids provide opportunities to create cell disease models, which can be studied to better understand the causes of diseases and determine possible treatment methods.

[0003] Conventional organoid chips are three-dimensional cell culture systems based on microfluidic chips. By constructing a microchannel system, organoids are placed in a suitable culture area, and then combined with a fluid pump and a control system, the culture medium is pushed into the chip to provide the necessary nutrients for the organoids. The channel design and manufacturing process based on microfluidic chips are relatively mature. However, currently, organoid chips generally have a culture function, but few detection functions. Detection of organoid-related indicators generally can only be carried out by sampling the culture medium, and the detected parameters are very limited and the operation process is cumbersome. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an organoid culture chip aiming at the deficiencies in the above-mentioned prior art.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: an organoid culture chip, comprising a pressing plate, an upper glass sheet, a flow channel layer sheet, a lower glass sheet, a biosensor, and a packaging bottom plate, which are arranged in sequence from top to bottom;

[0006] The flow channel layer sheet includes a layer sheet body and two culture units arranged side by side on the layer sheet body. Each culture unit includes N culture holes arranged in a line and penetrating the layer sheet body up and down, and a flow channel opened on the upper surface of the layer sheet body. The flow channel includes a liquid inlet main channel located in the middle of the layer sheet body, a liquid inlet branch channel communicated with the liquid inlet main channel, N distribution channels formed on the liquid inlet branch channel and respectively communicated with the inner sides of the N culture holes, N collection channels respectively communicated with the outer sides of the N culture holes, and a liquid discharge channel communicated with all the N collection channels. The two culture units are symmetrically arranged, and the flow channels of the two culture units share one liquid inlet main channel;

[0007] The biosensor is in the shape of a thin sheet, and its end has N detection chips that match the number and positions of the culture holes in the first culture unit among the two culture units. The N detection chips extend into the spaces below the N culture holes in the first culture unit one by one to monitor the biological parameters of the organoids in the N culture holes in this culture unit and provide reverse electrical stimulation.

[0008] Preferably, a baffle slot with the same depth as the converging flow channel is provided on the converging flow channel, and in the horizontal plane, the baffle slot is perpendicular to the converging flow channel, so that the baffle slot and the converging flow channel form a cross-shaped structure.

[0009] Preferably, a cuboid-shaped organoid baffle is fitted and inserted into the baffle slot. The organoid baffle is provided with a grid hole structure, and the grid hole structure has a size that does not allow organoids to pass through.

[0010] Preferably, a liquid inlet hole is provided in the middle of the upper glass sheet, two liquid outlet holes are provided on both sides of the liquid inlet hole, and a number of first air-permeable holes are provided.

[0011] Preferably, a liquid inlet pipe through-hole is provided in the middle of the pressing plate, two liquid outlet pipe through-holes are provided on both sides of the liquid inlet pipe through-hole, 2N upper observation holes corresponding to the 2N culture holes on the flow channel layer are provided, a number of second air-permeable holes are provided, and a number of screw through-holes are provided;

[0012] A liquid inlet pipe is inserted into the liquid inlet pipe through-hole, and the bottom of the liquid inlet pipe passes through the liquid inlet hole and extends into the main liquid inlet flow channel. A liquid discharge pipe is inserted into the liquid outlet pipe through-hole, and the bottom of the liquid discharge pipe passes through the liquid outlet hole and extends into the liquid discharge flow channel.

[0013] Preferably, the area of the bottom surface of the layer body below the first culture unit bulges downward to form a stepped surface, and rectangular grooves for accommodating the detection chips of the biosensor corresponding to the N culture holes in the first culture unit are provided on the bottom surface of the stepped surface.

[0014] Preferably, the thickness of the lower glass sheet is equal to the height of the bulge of the stepped surface. The lower glass sheet is arranged below the second culture unit to be flush with the stepped surface and can seal the bottoms of all the culture holes in the second culture unit;

[0015] A number of third air-permeable holes are provided on the lower glass sheet.

[0016] Preferably, the biosensor includes a T-shaped flat flexible PCB sheet, a pin header interface arranged at the end of the short side of the flexible PCB sheet, and the detection sheet arranged at the end of the long side of the flexible PCB sheet, the detection sheet and part of the long side of the flexible PCB sheet are inserted into the rectangular groove, and the detection sheet is located below the culture hole at the corresponding position and can close the bottom of the culture hole.

[0017] Preferably, the area on the packaging base plate directly below the first culture unit is provided with N rectangular bosses corresponding one-to-one to the N rectangular grooves, the area directly below the second culture unit is provided with N lower observation holes corresponding one-to-one to the N culture holes of the second culture unit, and the packaging base plate is also provided with a plurality of fourth air holes.

[0018] Preferably, the first air hole and the second air hole correspond in upper and lower positions, the third air hole corresponds in upper and lower positions to the fourth air hole on the packaging bottom plate, and the first air hole, the second air hole, the third air hole and the fourth air hole are all arranged away from the culture hole.

[0019] The beneficial effects of the present invention are:

[0020] The organoid culture chip provided by the present invention can detect various biological parameter indicators of the organoid in real time during the culture process by setting biosensors in some culture wells, and can provide reverse electrical stimulation to regulate the developmental function of the organoid. The biosensor of the present invention adopts a flat side outlet structure, which is convenient for wiring and does not affect the sealing of the culture chamber; by setting upper and lower light-transmitting structures in the other part of the culture wells, optical observation and Raman spectroscopy measurement can be performed in these culture wells;

[0021] The present invention can ensure the equivalence of the perfusion rate and the supply of the culture fluid in each culture well by setting an ingenious flow channel structure to match the culture unit; further, by setting an organoid baffle with a grid hole structure in the flow channel structure, the culture fluid can be normally flowed out of the culture well while blocking the organoid from flowing out of the culture well, thereby ensuring the effectiveness of the culture;

[0022] The culture chip is packaged using bonding and pressing processes, and the packaging method is reliable without leakage problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the exploded structure of the organoid culture chip of the present invention;

[0024] Figure 2 It is a schematic structural diagram of the front side of the flow channel layer of the present invention;

[0025] Figure 3 It is a structural schematic diagram of the baffle slot of the present invention;

[0026] Figure 4 Structural schematic diagram of the organoid baffle of the present invention;

[0027] Figure 5 Structural schematic diagram of the pressing plate of the present invention;

[0028] Figure 6 Structural schematic diagram of the upper glass sheet of the present invention;

[0029] Figure 7 Structural schematic diagram of the bottom surface of the flow channel layer sheet of the present invention;

[0030] Figure 8 Structural schematic diagram of the lower glass sheet of the present invention;

[0031] Figure 9 Structural schematic diagram of the biosensor of the present invention;

[0032] Figure 10 Structural schematic diagram of the encapsulation bottom plate of the present invention;

[0033] Figure 11 Overall assembly structural schematic diagram of the organoid culture chip of the present invention.

[0034] Explanation of reference numerals:

[0035] 1 - Pressing plate; 10 - Liquid inlet pipe through hole; 11 - Liquid outlet pipe through hole; 12 - Upper observation hole; 13 - Second air permeable hole; 14 - Screw through hole; 15 - Liquid inlet pipe; 16 - Drain pipe;

[0036] 2 - Upper glass sheet; 20 - Liquid inlet hole; 21 - Liquid outlet hole; 22 - First air permeable hole;

[0037] 3 - Flow channel layer sheet; 30 - Layer sheet body; 31 - First culture unit; 32 - Second culture unit; 33 - Culture hole; 34 - Flow channel; 35 - Organoid baffle; 340 - Main liquid inlet flow channel; 341 - Liquid inlet branch flow channel; 342 - Distribution flow channel; 343 - Collection flow channel; 344 - Drainage flow channel; 345 - Baffle groove; 350 - Grid hole structure; 36 - Step surface; 360 - Rectangular groove;

[0038] 4 - Lower glass sheet; 40 - Third air permeable hole;

[0039] 5 - Biosensor; 50 - Detection sheet; 51 - Flexible PCB sheet; 52 - Pin interface;

[0040] 6 - Encapsulation bottom plate; 60 - Rectangular boss; 61 - Lower observation hole; 62 - Fourth air permeable hole; 63 - Screw mounting hole;

[0041] 7 - Screw. Detailed implementation manners

[0042] The present invention will be further described in detail below in conjunction with embodiments, so that those skilled in the art can implement it with reference to the text of the specification.

[0043] It should be understood that terms such as "having", "comprising", and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0044] Embodiment 1

[0045] This embodiment provides an organoid culture chip, which includes a pressing plate 1, an upper glass sheet 2, a flow channel layer sheet 3, a lower glass sheet 4, a biosensor 5, and a packaging bottom plate 6 arranged in sequence from top to bottom;

[0046] The flow channel layer sheet 3 includes a layer sheet body 30 and two culture units arranged side by side on the layer sheet body 30. The culture unit includes N culture holes 33 arranged in a line and penetrating the layer sheet body 30 up and down, and a flow channel 34 opened on the upper surface of the layer sheet body 30. The flow channel 34 includes a liquid inlet main channel 340 located in the middle of the layer sheet body 30, a liquid inlet branch channel 341 communicating with the liquid inlet main channel 340, N distribution channels 342 formed on the liquid inlet branch channel 341 and respectively communicating with the inner sides of the N culture holes 33, N collecting channels 343 respectively communicating with the outer sides of the N culture holes 33, and a liquid discharge channel 344 communicating with all the N collecting channels. The two culture units are symmetrically arranged, and the flow channels 34 of the two culture units share a liquid inlet main channel 340;

[0047] The biosensor 5 is in the shape of a thin sheet, and its end has N detection sheets 50 that match the number and position of the culture holes 33 in the first culture unit 31 of the two culture units. The N detection sheets 50 respectively extend into the lower parts of the N culture holes 33 in the first culture unit 31 to monitor biological parameters (such as electrophysiological signals, metabolite indicators, pH levels, cell mechanical parameters, etc.) of the organoids in the N culture holes 33 of this culture unit and provide reverse electrical stimulation.

[0048] In this embodiment, N = 4 is taken as an example for illustration. The eight culture wells 33 in the two culture units are arranged in a 4×2 two-row layout. The main liquid inlet channel 340 is located at the center and is shared by the two culture units. Four are correspondingly arranged in each culture unit. Four distribution channels 342 are connected in parallel on the branch liquid inlet channel 341 to achieve uniform liquid inlet distribution for the four culture wells 33. One collecting channel 343 is symmetrically arranged on each culture well 33 with respect to the distribution channel 342. The four collecting channels 343 finally converge into one liquid discharge channel 344. The culture solution entering through the main liquid inlet channel 340 is evenly distributed to each culture well 33 through the main liquid inlet channel 340. The culture solution in the culture well 33 then enters the collecting channel 343 and converges into the liquid discharge channel 344 and is finally discharged, ensuring the equivalence of the perfusion rate and the culture solution supply for each culture well 33. In a preferred embodiment, the layer body 30 can be made of PDMS material.

[0049] In a preferred embodiment, a baffle groove 345 with the same depth as the collecting channel 343 is formed on the collecting channel 343, and in the horizontal plane, the baffle groove 345 is perpendicular to the collecting channel 343, so that the baffle groove 345 and the collecting channel 343 form a cross-shaped structure. A rectangular parallelepiped-shaped organoid baffle 35 is inserted into the baffle groove 345 in a matching manner. A grid hole structure 350 is formed on the organoid baffle 35. The grid hole structure 350 has a size that does not allow organoids to pass through, such as 0.1 - 0.2 mm. In this way, the culture solution can flow out normally through the grid holes, while blocking the escape of organoids from the chip, ensuring the effectiveness of the culture.

[0050] In this embodiment, the upper glass sheet 2 is attached to the upper surface of the flow channel layer sheet 3 to seal the upper part of the culture well 33. An inlet hole 20 located in the middle, two outlet holes 21 located on both sides of the inlet hole 20, and a number of first air holes 22 are formed on the upper glass sheet 2. A number of first air holes 22 are formed around each culture well 33. The PDMS of the flow channel 34 layer is a breathable material. The CO2 culture gas in the culture environment can be dissolved into the culture solution through the air holes passing through the flow channel 34 layer to control the pH level of the culture solution.

[0051] In this embodiment, a through hole 10 for the inlet pipe 15 located in the middle, two through holes 11 for the outlet pipe located on both sides of the through hole 10 for the inlet pipe 15, 2N upper observation holes 12 corresponding to the 2N culture wells 33 on the flow channel layer sheet 3 one by one, a number of second air holes 13, and a number of screw through holes 14 are formed on the pressing plate 1.

[0052] A liquid inlet pipe 15 is inserted into the through-hole 10, and the bottom of the liquid inlet pipe 15 passes through the liquid inlet hole 20 and extends into the main liquid inlet channel 340. A liquid discharge pipe 16 is inserted into the through-hole 11 of the liquid discharge pipe, and the bottom of the liquid discharge pipe 16 passes through the liquid discharge hole 21 and extends into the liquid discharge channel 344. In a preferred embodiment, the material of the pressing plate 1 can be a transparent engineering plastic, such as transparent PMMA, which is convenient for observing the flow channel 34 and the organoid culture situation, and at the same time has a certain strength and toughness, and will not be crushed when the screw is tightened.

[0053] In this embodiment, the bottom surface of the laminate body 30 in the area below the first culture unit 31 bulges downward to form a stepped surface 36, and N rectangular grooves 360 for accommodating the detection chips 50 of the biosensors 5 are formed in the bottom surface of the stepped surface 36 and are in one-to-one correspondence and communication with the N culture holes 33 in the first culture unit 31.

[0054] The thickness of the lower glass sheet 4 is equal to the height of the protrusion of the stepped surface 36. The lower glass sheet 4 is arranged below the second culture unit 32 to be flush with the stepped surface 36 to make up the height difference and can seal the bottoms of all the culture holes 33 in the second culture unit 32; a plurality of third air-permeable holes 40 are formed in the lower glass sheet 4.

[0055] In this embodiment, the biosensor 5 includes a flat flexible PCB sheet 51 in a T shape, a pin header interface 52 arranged at the end of the short side of the flexible PCB sheet 51, and a detection chip 50 arranged at the end of the long side of the flexible PCB sheet 51. The detection chip 50 is a rectangular sheet with a certain thickness. The rectangular sheet is a Si substrate, the upper part is a detection surface facing the culture hole 33, and the lower part is bonded to the flexible PCB sheet 51; the pin header interface 52 extends outwards and can be connected to an external connector for transmitting signals out;

[0056] The width of the detection chip 50 matches the width of the rectangular groove 360. The long sides of the detection chip 50 and part of the flexible PCB sheet 51 are inserted into the rectangular groove 360, and the detection chip 50 is located below the corresponding culture hole 33 and can seal the bottom of the culture hole 33.

[0057] In this embodiment, N rectangular bosses 60 corresponding to the N rectangular grooves 360 are arranged in the area of the encapsulation bottom plate 6 directly below the first culture unit 31, and N lower observation holes 61 corresponding to the N culture holes 33 of the second culture unit 32 are formed in the area directly below the second culture unit 32. A plurality of fourth air-permeable holes 62 are also formed in the encapsulation bottom plate 6.

[0058] The height of the rectangular boss 60 is matched according to the thickness of the detection surface of different biosensors 5. When the surface of the encapsulation bottom plate 6 is attached to the back surface of the flow channel 34 layer, the gap between the rectangular boss 60 and the rectangular groove 360 just accommodates the thickness of the biosensing detection chip 50, so as to realize the sealing of the flow channel 34. The material of the encapsulation bottom plate 6 can be selected from materials with higher stiffness, such as stainless steel.

[0059] The upper and lower positions of the first air vent 22 and the second air vent 13 correspond to each other, and the upper and lower positions of the third air vent 40 and the fourth air vent 62 on the encapsulation bottom plate 6 correspond to each other. Moreover, the first air vent 22, the second air vent 13, the third air vent 40, and the fourth air vent 62 are all arranged avoiding the culture holes 33.

[0060] Multiple screw mounting holes 63 are provided in the middle and the periphery of the encapsulation bottom plate 6. During assembly, the upper glass sheet 2, the flow channel 34 layer, and the lower glass sheet 4 are first keyed into an intermediate component, and then the biosensor 5 is placed in the rectangular groove 360. Several screws 7 pass through the screw through holes 14 on the pressing plate 1 and are inserted into the screw mounting holes 63 on the encapsulation bottom plate 6 to press and fasten the intermediate component, ensuring the sealing effect of the flow channel 34. The effect after encapsulation is as Figure 11 shown. The pin interface 52 for biosensing can extend horizontally from the side for external wiring.

[0061] The biosensor 5 is installed at the bottom of the culture hole 33 of the first culture unit 31, and can monitor the development-related parameters of the organoids in real time during the culture, such as electrophysiological signals, metabolite indicators, pH levels, cell mechanics parameters, etc. At the same time, it can also provide reverse electrical stimulation to regulate the development function of the organoids; the culture holes 33 of the second culture unit 32 are transparent up and down and have observation windows, which can be used for optical observation and Raman spectroscopy measurement.

[0062] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. An organoid culture chip, characterized in that, It includes a pressing plate, an upper glass sheet, a flow channel layer sheet, a lower glass sheet, a biosensor and a packaging bottom plate which are arranged in sequence from top to bottom; The flow channel layer sheet includes a layer sheet body and two culture units arranged side by side on the layer sheet body. Each culture unit includes N culture holes arranged in a line and penetrating through the layer sheet body vertically, and a flow channel formed on the upper surface of the layer sheet body. The flow channel includes a liquid inlet main channel located in the middle of the layer sheet body, a liquid inlet branch channel communicated with the liquid inlet main channel, N distribution channels formed on the liquid inlet branch channel and respectively communicated with the inner sides of the N culture holes, N collecting channels respectively communicated with the outer sides of the N culture holes, and a liquid discharge channel communicated with all the N collecting channels. The two culture units are symmetrically arranged and the flow channels of the two culture units share one liquid inlet main channel; The biosensor is in the shape of a thin sheet, and its end has N detection sheets which match the number and positions of the culture holes in the first culture unit of the two culture units. The N detection sheets respectively extend into the lower parts of the N culture holes in the first culture unit to monitor the biological parameters of the organoids in the N culture holes in this culture unit and provide reverse electrical stimulation.

2. The organoid culture chip according to claim 1, wherein, A baffle slot with the same depth as the collecting channel is formed on the collecting channel, and in the horizontal plane, the baffle slot is perpendicular to the collecting channel, so that the baffle slot and the collecting channel form a cross-shaped structure.

3. The organoid culture chip according to claim 2, characterized in that, A cuboid-shaped organoid baffle is inserted into the baffle slot in a matching manner. The organoid baffle is provided with a grid hole structure, and the grid hole structure has a size that does not allow organoids to pass through.

4. The organoid culture chip according to claim 1, wherein, An inlet hole is formed in the middle of the upper glass sheet, two outlet holes are formed on both sides of the inlet hole, and a number of first air holes are formed.

5. The organoid culture chip according to claim 4, wherein A liquid inlet pipe through hole is formed in the middle of the pressing plate, two liquid outlet pipe through holes are formed on both sides of the liquid inlet pipe through hole, 2N upper observation holes corresponding to the 2N culture holes on the flow channel layer sheet, a number of second air holes and a number of screw through holes are formed; A liquid inlet pipe is inserted into the liquid inlet pipe through hole, and the bottom of the liquid inlet pipe passes through the inlet hole and extends into the liquid inlet main channel. A liquid discharge pipe is inserted into the liquid outlet pipe through hole, and the bottom of the liquid discharge pipe passes through the outlet hole and extends into the liquid discharge channel.

6. The organoid culture chip according to claim 5, wherein The area of the bottom surface of the layer sheet body below the first culture unit bulges downward to form a step surface, and N rectangular grooves for accommodating the detection sheets of the biosensor are formed on the bottom surface of the step surface and respectively communicated with the N culture holes in the first culture unit.

7. The organoid culture chip according to claim 6, wherein, The thickness of the lower glass sheet is equal to the protruding height of the step surface. The lower glass sheet is arranged below the second culture unit of the two culture units to be flush with the step surface and can seal the bottoms of all the culture holes in the second culture unit; A number of third air holes are formed on the lower glass sheet.

8. The organoid culture chip according to claim 7, wherein, The biosensor includes a flat flexible PCB sheet in a T shape, a pin header interface provided at the end of the short side of the flexible PCB sheet, and the detection sheet provided at the end of the long side of the flexible PCB sheet. The long side of the detection sheet and a part of the flexible PCB sheet are inserted into the rectangular groove, and the detection sheet is located below the culture well at the corresponding position and can seal the bottom of the culture well.

9. The organoid culture chip according to claim 8, wherein In the area of the encapsulation bottom plate directly below the first culture unit, N rectangular bosses corresponding to the N rectangular grooves one by one are provided. In the area directly below the second culture unit, N lower observation holes corresponding to the N culture wells of the second culture unit one by one are provided. A plurality of fourth ventilation holes are also provided on the encapsulation bottom plate.

10. The organoid culture chip according to claim 9, wherein The first ventilation hole and the second ventilation hole are vertically corresponding to each other. The third ventilation hole is vertically corresponding to the fourth ventilation hole on the encapsulation bottom plate. Moreover, the first ventilation hole, the second ventilation hole, the third ventilation hole, and the fourth ventilation hole are all arranged to avoid the culture wells.

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