Organoid culture device

Abstract

The present invention discloses an organoid culture device, comprising: a culture chamber, which includes a base assembly and an upper cover assembly rotatably connected to the base assembly. The upper cover assembly includes an upper cover, an upper heating film, an upper window, a temperature and humidity sensor, and a locking mechanism provided on the upper cover. The base assembly includes a base, a lower heating film, a temperature sensor, a humidifying dish, and a lower window provided on the base; the locking mechanism is used to cooperate with the base to lock the upper cover on the base; and a culture chip, which is arranged inside the culture chamber and is located between the upper window and the lower window. The organoid culture device provided by the present invention can realize the integration of culture, observation, and biosensing detection. By providing a biosensor, relevant parameters related to the development of organoids can be detected in real time during the culture, and reverse electrical stimulation can be provided to regulate the development function of organoids; moreover, the biosensor of the present invention adopts a flat side wire outlet structure, which is convenient for wiring and does not affect the sealing of the culture chamber.

Description

Technical Field

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

[0002] In vitro cell culture is the most commonly used and important research method in life science research. For a long time, 2D cell culture has occupied an important position in the culture technology, but more and more evidence shows that it has various drawbacks. 3D culture is to co-culture stem cells, growth factors and artificial matrix skeletons to simulate the microenvironment of in vivo cell growth. Organoid culture is a 3D cell culture system aiming to construct a cell model closer to the characteristics of the source tissue or organ.

[0003] Organoids belong to three-dimensional (3D) cell cultures. The basic environments necessary for their long-term culture and function maintenance mainly include: (a) temperature conditions, ensuring that the organoids grow at a preset temperature with an error controlled within ±0.2 °C; (b) gas conditions, automatically mixing CO2, O2 and N2 in a set ratio according to different stages and requirements of organoid development; (c) humidity conditions, the culture environment should ensure high humidity requirements to reduce the impact of changes in the osmotic pressure of the culture medium on the organ; (d) fluid conditions, realizing automatic supply of the culture medium, timely removal of metabolites, and at the same time providing the shear force environment required for organoid development. In addition, external stimuli such as electrical stimulation and light stimulation can be applied during the culture process of organoids to further regulate and maintain their behaviors and functions.

[0004] Existing organoid culture devices are discrete. The incubator uses a conventional CO2 incubator, and the culture dish uses a conventional well plate type for culture. The culture process of organoids is relatively long, reaching several months or even longer. It is necessary to regularly take out the culture dish from the incubator for liquid change operations. Moreover, the detection of organoids usually adopts sampling or offline methods and is operated on external dedicated instruments, unable to achieve integrated and automated operations of culture, observation and detection. The operation process is cumbersome and easily affects the normal development of organoids. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an organoid culture device in view of the above deficiencies in the prior art.

[0006] To solve the above technical problem, the technical solution adopted by the present invention is: an organoid culture device, comprising:

[0007] A culture chamber, comprising a base assembly and an upper cover assembly rotatably connected to the base assembly, wherein the upper cover assembly comprises an upper cover and an upper heating film, an upper window, a temperature and humidity sensor and a locking mechanism arranged on the upper cover, and the base assembly comprises a base and a lower heating film, a temperature sensor, a humidifying dish and a lower window arranged on the base; the locking mechanism is used to cooperate with the base to realize the locking of the upper cover on the base;

[0008] And a culture chip, which is arranged inside the culture chamber and between the upper window and the lower window.

[0009] Preferably, the surface and bottom surface of the upper window are provided with grooves, each of the grooves is provided with a glass sheet, and there is a gap between the two glass sheets in the upper window to form an air layer;

[0010] The surface and the bottom surface of the lower window are also provided with grooves, each of which is provided with a glass sheet, and there is a gap between the two glass sheets in the lower window to form an air layer.

[0011] Preferably, the locking mechanism is arranged at the end of the upper cover, and the locking mechanism comprises a swing block rotatably connected to the end of the upper cover through a pin shaft, a paddle connected to the outer end of the swing block, a lock hook connected to the lower part of the swing block, a tension spring connected between the end of the upper cover and the swing block, a limit handle connected to the end of the upper cover and located above the paddle, and a positioning pin connected to the bottom of the upper cover, and a positioning hole for the positioning pin to be inserted into is provided on the base;

[0012] A barb for cooperating with the lock hook is provided at a corresponding position on the base below the lock hook;

[0013] A waist hole is provided at the lower part of the swing block, and the locking hook is connected to the waist hole by screws, so that the height of the locking hook in the vertical direction can be adjusted;

[0014] An annular sealing ring is arranged on the bottom edge of the upper cover.

[0015] Preferably, two limit blocks are provided along the diagonal direction on the periphery of the lower window to limit the position of the culture chip.

[0016] Preferably, a gas interface pipe is provided on the side of the humidifying dish, and the gas outlet end of the gas interface pipe goes deep into the bottom of the humidifying dish, so that the gas discharged from the gas interface pipe is humidified by the water in the humidifying dish and then input into the culture chip;

[0017] A plurality of liquid interface tubes are also arranged between the humidifying dish and the lower window to realize the perfusion of the culture liquid in the culture chip.

[0018] Preferably, the culture chip 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;

[0019] The flow channel layer sheet includes a sheet body and two culture units arranged side by side on the sheet body. The culture unit includes N culture holes arranged in a line and penetrating the sheet body vertically, and a flow channel formed on the upper surface of the sheet body. The flow channel includes a liquid inlet main channel located in the middle of the sheet body, a liquid inlet branch channel communicating with the liquid inlet main channel, N distribution channels formed on the liquid inlet branch channel and respectively communicating with the inner sides of the N culture holes one by one, N collection channels respectively communicating with the outer sides of the N culture holes one by one, and a liquid discharge channel communicating 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;

[0020] The biosensor is in the shape of a thin sheet, and its end has N detection pieces that match the number and position of the culture holes in the first culture unit among the two culture units. The N detection pieces 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.

[0021] Preferably, 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 plurality of first air holes are formed.

[0022] Preferably, 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 plurality of second air holes, and a plurality of screw through holes;

[0023] 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. The liquid inlet pipe and the liquid discharge pipe are respectively connected to the corresponding liquid path interface pipes.

[0024] Preferably, the area of the bottom surface of the sheet body below the first culture unit bulges downward to form a stepped surface, and N rectangular grooves for accommodating the detection pieces of the biosensor are formed on the bottom surface of the stepped surface and respectively communicate with the N culture holes in the first culture unit one by one.

[0025] Preferably, the thickness of the lower glass sheet is equal to the height of the protrusion on the stepped surface. The lower glass sheet is disposed below the second culture unit in the two culture units to be flush with the stepped surface and can seal the bottoms of all the culture holes in the second culture unit.

[0026] A plurality of third air-permeable holes are formed in the lower glass sheet.

[0027] Preferably, the biosensor includes a flat flexible PCB sheet in a T shape, a pin header interface disposed at the end of the short side of the flexible PCB sheet, and the detection sheet disposed 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 disposed below the culture hole at the corresponding position and can seal the bottom of the culture hole.

[0028] Preferably, in the area of the encapsulation bottom plate directly below the first culture unit, N rectangular bosses corresponding to the N rectangular grooves are provided. In the area directly below the second culture unit, N lower observation holes corresponding to the N culture holes of the second culture unit are formed. A plurality of fourth air-permeable holes are also formed in the encapsulation bottom plate.

[0029] Preferably, the first air-permeable hole and the second air-permeable hole are vertically corresponding to each other. The third air-permeable hole and the fourth air-permeable hole on the encapsulation bottom plate are vertically corresponding to each other. Moreover, the first air-permeable hole, the second air-permeable hole, the third air-permeable hole, and the fourth air-permeable hole are all arranged to avoid the culture holes.

[0030] The beneficial effects of the present invention are as follows:

[0031] 1) The organoid culture device provided by the present invention can realize the integration of culture, observation, and biosensing detection. By providing a biosensor, the development-related parameters of the organoids can be detected in real time during the culture period, and reverse electrical stimulation can be provided to regulate the development function of the organoids. Moreover, the biosensor of the present invention adopts a flat side-out wiring structure, which is convenient for wiring and does not affect the sealing of the culture chamber.

[0032] 2) The culture chamber of the present invention has the functions of temperature control and humidity control and can realize liquid supply and gas supply operations.

[0033] 3) By providing a locking mechanism, the present invention can realize the convenient closing and unlocking of the upper cover and the base lock of the culture chamber, and the sealing effect is adjustable.

[0034] 4) In the present invention, the upper and lower observation windows adopt a double-layer glass design, which can effectively avoid the condensation and fogging situation.

[0035] 5) The culture chip of the present invention is encapsulated by a bonding and pressing process, and the encapsulation method is reliable and there is no liquid leakage problem.

[0036] 6) The culture chip of the present invention is provided with an observation window at the culture well position, facilitating optical observation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic structural diagram of the organoid culture device of the present invention;

[0038] Figure 2 is a schematic structural diagram of the upper cover assembly of the present invention;

[0039] Figure 3 is a schematic structural diagram of the upper window of the upper cover of the present invention;

[0040] Figure 4 is a sectional view of the upper cover assembly of the present invention;

[0041] Figure 5 is a sectional view of the upper window of the present invention;

[0042] Figure 6 is a partially enlarged schematic structural diagram of the locking mechanism of the present invention;

[0043] Figure 7 is a schematic structural diagram of the locking mechanism of the present invention from the bottom view perspective;

[0044] Figure 8 is a schematic structural diagram of the base assembly of the present invention;

[0045] Figure 9 is a sectional view of the base assembly of the present invention;

[0046] Figure 10 is a sectional view of the lower window of the present invention;

[0047] Figure 11 is a sectional view at the humidifying dish of the present invention;

[0048] Figure 12 is an exploded schematic structural diagram of the culture chip of the present invention;

[0049] Figure 13 is a schematic structural diagram of the front side of the flow channel layer sheet of the present invention;

[0050] Figure 14 is a schematic structural diagram of the upper glass sheet of the present invention;

[0051] Figure 15 is a schematic structural diagram of the bottom side of the flow channel layer sheet of the present invention;

[0052] Figure 16 is a schematic structural diagram of the lower glass sheet of the present invention;

[0053] Figure 17 is a schematic structural diagram of the biosensor of the present invention;

[0054] Figure 18 It is a structural schematic diagram of the packaging base plate of the present invention;

[0055] Figure 19 It is a schematic diagram of the overall assembly structure of the culture chip of the present invention.

[0056] Description of reference numerals:

[0057] 1—culture chamber; 10—groove; 11—glass slide;

[0058] 2—upper cover assembly; 20—upper cover; 21—upper heating film; 22—upper window; 23—temperature and humidity sensor;

[0059] 24—locking mechanism; 240—swing block; 241—pin shaft; 242—pick; 243—lock hook; 244—tension spring; 245—limiting handle; 246—positioning pin; 247—waist hole;

[0060] 25—annular sealing ring;

[0061] 3—base assembly; 30—base; 31—lower heating film; 32—temperature sensor; 33—humidifying dish; 34—lower window; 35—positioning hole; 36—barb; 37—limiting block; 38—gas interface pipe; 39—liquid interface pipe; 300—hinge;

[0062] 4—Cultivate chip;

[0063] 41—upper glass sheet; 410—liquid inlet hole; 411—liquid outlet hole; 412—first vent hole; 413—screw hole; 414—liquid inlet pipe; 415—liquid outlet pipe;

[0064] 42—channel sheet; 420—sheet body; 421—first culture unit; 422—second culture unit; 423—culture hole; 424—channel; 425—step surface; 4240—main inlet channel; 4241—branch inlet channel; 4242—distribution channel; 4243—collection channel; 4244—drainage channel; 4250—rectangular groove;

[0065] 43—lower glass sheet; 430—second vent hole;

[0066] 44—biosensor; 440—detection sheet; 441—flexible PCB sheet; 442—pin header interface;

[0067] 45—packaging bottom plate; 450—rectangular boss; 451—lower observation hole; 452—third vent hole; 453—screw mounting hole;

[0068] 46—Screw. DETAILED DESCRIPTION

[0069] 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.

[0070] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0071] Embodiment 1

[0072] This embodiment provides an organoid culture device, including:

[0073] A culture chamber 1, which includes a base assembly 3 and an upper cover assembly 2 rotatably connected to the base assembly 3. The upper cover assembly 2 includes an upper cover 20, an upper heating film 21, an upper window 22, a temperature and humidity sensor 23, and a locking mechanism 24 provided on the upper cover 20. The base assembly 3 includes a base 30, a lower heating film 31, a temperature sensor 32, a humidifying dish 33, and a lower window 34 provided on the base 30. The locking mechanism 24 is used to cooperate with the base 30 to lock the upper cover 20 on the base 30.

[0074] And a culture chip 4, which is arranged inside the culture chamber 1 and is located between the upper window 22 and the lower window 34. The culture chip 4 provides a space for organoid culture inside.

[0075] In this embodiment, a hinge 300 is provided on the base 30, and the upper cover 20 is rotatably connected to the base 30 through the hinge 300.

[0076] In this embodiment, an upper cover window is opened at the top of the upper cover 20. The upper cover window is round hole-shaped and arranged in a line, corresponding to the position of the culture holes 423 of the culture chip 4 for convenient optical observation. Grooves 10 are provided on both the surface and the bottom surface of the upper window 22, and a glass sheet 11 is arranged in each groove 10 for sealing. There is a gap between the two glass sheets 11 in the upper window 22 to form an air layer, which can achieve a good heat preservation effect. When there is only one glass sheet 11, the temperature of the glass sheet 11 is lower than the temperature of the gas in the culture chamber 1, and the humid and hot gas in the culture chamber 1 will condense and fog when it encounters the cold glass, affecting optical observation. The double-layer glass is heat-insulated through the intermediate air layer to avoid this situation.

[0077] Similarly, grooves 10 are provided on both the surface and the bottom surface of the lower window 34, and a glass sheet 11 is arranged in each groove 10, and there is a gap between the two glass sheets 11 in the lower window 34 to form an air layer.

[0078] The upper heating film 21 is pasted on the top of the upper cover 20, and the temperature and humidity sensor 23 detects the temperature at the position of the upper cover 20 and cooperates with the upper heating film 21 to control the temperature of the upper cover 20.

[0079] In this embodiment, the locking mechanism 24 is arranged at the end of the upper cover 20. The locking mechanism 24 includes a swing block 240 rotatably connected to the end of the upper cover 20 through a pin shaft 241, a dial 242 connected to the outer end of the swing block 240, a locking hook 243 connected to the lower part of the swing block 240, a tension spring 244 connected between the end of the upper cover 20 and the swing block 240, a limit handle 245 connected to the end of the upper cover 20 and above the dial 242, and a positioning pin 246 connected to the bottom of the upper cover 20. A positioning hole 35 for the positioning pin 246 to be inserted into is formed on the base 30.

[0080] At a corresponding position on the base 30 below the locking hook 243, an inverted hook 36 for cooperating with the locking hook 243 is provided; the connection point of the tension spring 244 on the swing block 240 is located between the locking hook 243 and the pin shaft 241. Under the action of the tension spring 244, the locking hook 243 is pressed against the end direction of the upper cover 20 ( Figure 6 the left side in the figure), so as to hook the inverted hook 36 on the base 30; the dial 242 is fixed on the swing block 240 in the direction opposite to the tension spring 244. When the dial 242 is pushed upward and extruded, the swing block 240 will rotate counterclockwise against the acting force of the tension spring 244, and the locking hook 243 will disengage from the inverted hook 36 to release the locking, so that the upper cover 20 can be opened; the limit handle 245 is arranged above the dial 242 at a certain interval, and can control the swing angle of the swing block 240. The upper cover 20 and the base 30 are respectively designed with a positioning pin 246 and a positioning hole 35 to achieve rapid positioning. When the upper cover 20 is buckled in the base 30, the positioning pin 246 is inserted into the positioning hole 35 in cooperation, and then locked by the locking mechanism 24.

[0081] A waist-shaped hole 247 is formed in the lower part of the swing block 240, and the locking hook 243 is connected to the waist-shaped hole 247 through a screw 46, so that the height of the locking hook 243 in the vertical direction can be adjusted; an annular sealing ring 25 is arranged on the bottom edge of the upper cover 20. Thus, the pre-tightening amount of the annular sealing ring 25 of the culture chamber 1 can be adjusted to improve the sealing performance.

[0082] In this embodiment, two limit blocks 37 are arranged along the diagonal direction on the periphery of the lower window 34 to limit the position of the culture chip 4.

[0083] In this embodiment, two culture stations are arranged in the culture chamber 1, and two culture chips 4 can be arranged simultaneously; an upper window 22 and a lower window 34 are arranged corresponding to each other up and down for each culture station, and two limit blocks 37 are arranged corresponding to each culture station.

[0084] An air passage interface pipe 38 is provided on the side of the humidifying dish 33, and the air outlet end of the air passage interface pipe 38 extends deep into the bottom of the humidifying dish 33, so that the gas discharged from the air passage interface pipe 38 is humidified by the water in the humidifying dish 33 to form bubbles and escape, which can humidify the culture chamber 1. By adjusting the gas flow rate, the humidity level can be changed. The humidity situation in the culture chamber 1 can be monitored by the temperature and humidity sensor 23 of the upper cover assembly 2.

[0085] The lower heating film 31 is pasted on the back of the base 30, and the temperature sensor 32 detects the temperature of the base 30 and cooperates with the lower heating film 31 to control the temperature of the base 30.

[0086] A number of liquid passage interface pipes 39 are also provided between the humidifying dish 33 and the lower window 34 to realize the perfusion of the culture solution in the culture chip 4.

[0087] Embodiment 2

[0088] As a further improvement based on Embodiment 1, this embodiment also provides a culture chip 4, which includes a pressing plate, an upper glass sheet 41, a flow channel layer sheet 42, a lower glass sheet 43, a biosensor 44 and a packaging bottom plate 45 arranged in sequence from top to bottom;

[0089] The flow channel layer sheet 42 includes a layer sheet body 420 and two culture units arranged side by side on the layer sheet body 420. The culture unit includes N culture holes 423 arranged in a row and penetrating through the layer sheet body 420 up and down, and a flow channel 424 opened on the upper surface of the layer sheet body 420. The flow channel 424 includes a liquid inlet main channel 4240 located in the middle of the layer sheet body 420, a liquid inlet branch channel 4241 communicated with the liquid inlet main channel 4240, N distribution channels 4242 formed on the liquid inlet branch channel 4241 and corresponding to and communicated with the inner sides of the N culture holes 423 respectively, N collecting channels 4243 corresponding to and communicated with the outer sides of the N culture holes 423 respectively, and a liquid discharge channel 4244 communicated with all the N collecting channels. The two culture units are symmetrically arranged and the flow channels of the two culture units share a liquid inlet main channel 4240;

[0090] The biosensor 44 is in the shape of a thin sheet, and its end has N detection pieces 440 that match the number and position of the culture holes 423 in the first culture unit 421 of the two culture units. The N detection pieces 440 extend into the lower part of the N culture holes 423 in the first culture unit 421 respectively to monitor the biological parameters of the organoids in the N culture holes 423 in this culture unit and provide reverse electrical stimulation.

[0091] In this embodiment, N = 4 is taken as an example for illustration. The 8 culture wells 423 in the two culture units are arranged in a 4×2 two-row layout. The main liquid inlet channel 4240 is located at the center and is shared by the two culture units. There are 4 corresponding ones in each culture unit. 4 distribution channels 4242 are arranged in parallel on the branch liquid inlet channel 4241 to achieve uniform liquid inlet distribution for the 4 culture wells 423. One collecting channel 4243 is symmetrically arranged on each culture well 423 with respect to the distribution channel 4242. The 4 collecting channels 4243 finally converge into 1 drainage channel 4244. The culture solution entering through the main liquid inlet channel 4240 is evenly distributed to each culture well 423 through the main liquid inlet channel 4240. The culture solution in the culture well 423 then enters the collecting channel 4243 and converges into the drainage channel 4244 and is finally discharged, ensuring the equivalence of the perfusion rate and the culture solution supply for each culture well 423. In a preferred embodiment, the layer body 420 can be made of PDMS material.

[0092] In this embodiment, the upper glass sheet 41 is attached to the upper surface of the flow channel layer sheet 42 to seal the upper part of the culture well 423. An inlet hole 410 located in the middle, two outlet holes 411 located on both sides of the inlet hole 410, and several first ventilation holes 412 are provided on the upper glass sheet 41. The several first ventilation holes 412 are arranged around the vicinity of each culture well 423. The flow channel layer PDMS is a breathable material, and the CO2 culture gas in the culture environment can pass through the ventilation holes and dissolve into the culture solution through the flow channel layer to control the pH level of the culture solution.

[0093] In this embodiment, a through hole for the inlet pipe 414 located in the middle, two through holes for the outlet pipes located on both sides of the through hole for the inlet pipe 414, 2N upper observation holes corresponding to the 2N culture wells 423 on the flow channel layer sheet 42, several second ventilation holes 430, and several screw through holes 413 are provided on the pressing plate;

[0094] An inlet pipe 414 whose bottom passes through the inlet hole 410 and extends into the main liquid inlet channel 4240 is inserted into the through hole for the inlet pipe 414, and a drainage pipe 415 whose bottom passes through the outlet hole 411 and extends into the drainage channel 4244 is inserted into the through hole for the outlet pipe. The inlet pipe 414 and the drainage pipe 415 are respectively connected to the corresponding liquid path interface pipes 39 to achieve the filling and discharging of the culture solution. In a preferred embodiment, the pressing plate material can be made of a transparent engineering plastic, such as transparent PMMA, which is convenient for observing the flow channel and the organoid culture situation, and at the same time has a certain strength and toughness and will not be crushed when fastened by screws.

[0095] In this embodiment, the bottom surface of the laminate body 420 bulges downward in the area below the first culture unit 421 to form a stepped surface 425, and four rectangular grooves 4250 for accommodating the detection pieces 440 of the biosensors 44 are formed at the bottom surface of the stepped surface 425 and are in one-to-one correspondence and communication with the four culture holes 423 in the first culture unit 421.

[0096] The thickness of the lower glass sheet 43 is equal to the protruding height of the stepped surface 425. The lower glass sheet 43 is disposed below the second culture unit 422 to be flush with the stepped surface 425 to make up the height difference and can seal the bottoms of all the culture holes 423 in the second culture unit 422; a plurality of third air-permeable holes 452 are formed in the lower glass sheet 43.

[0097] In this embodiment, the biosensor 44 includes a flat flexible PCB sheet 441 in a T shape, a pin header interface 442 disposed at the end of the short side of the flexible PCB sheet 441, and a detection piece 440 disposed at the end of the long side of the flexible PCB sheet 441. The detection piece 440 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 423, and the lower part is bonded to the flexible PCB sheet 441; the pin header interface 442 extends outwards and can be connected to an external connector for transmitting signals out.

[0098] The width of the detection piece 440 matches the width of the rectangular groove 4250. The long sides of the detection piece 440 and a part of the flexible PCB sheet 441 are inserted into the rectangular groove 4250, and the detection piece 440 is located below the corresponding culture hole 423 and can seal the bottom of the culture hole 423.

[0099] In this embodiment, N rectangular bosses 450 corresponding to the N rectangular grooves 4250 are provided in the area of the encapsulation bottom plate 45 directly below the first culture unit 421, and N lower observation holes 451 corresponding to the N culture holes 423 of the second culture unit 422 are formed in the area directly below the second culture unit 422. A plurality of fourth air-permeable holes are also formed in the encapsulation bottom plate 45.

[0100] The height of the rectangular boss 450 is matched according to the thickness of the detection surface of different biosensors 44, so as to ensure that when the surface of the encapsulation bottom plate 45 is attached to the back surface of the flow channel layer, the gap between the rectangular boss 450 and the rectangular groove 4250 just accommodates the thickness of the biosensor detection piece 440, thereby realizing flow channel sealing. The material of the encapsulation bottom plate 45 can be selected from materials with relatively high stiffness, such as stainless steel.

[0101] The first air-permeable hole 412 and the second air-permeable hole 430 are vertically corresponding to each other, the third air-permeable hole 452 and the fourth air-permeable hole on the encapsulation bottom plate 45 are vertically corresponding to each other, and the first air-permeable hole 412, the second air-permeable hole 430, the third air-permeable hole 452, and the fourth air-permeable hole are all arranged avoiding the culture holes 423.

[0102] Multiple screw mounting holes 453 are provided in the middle and periphery of the encapsulation bottom plate 45. During assembly, the upper glass sheet 41, the flow channel layer, and the lower glass sheet 43 are first keyed together into an intermediate component, and then the biosensor 44 is placed in the rectangular groove 4250. Several screws 46 pass through the screw through holes 413 on the pressing plate and are inserted into the screw mounting holes 453 on the encapsulation bottom plate 45 to press and fasten the intermediate component, ensuring the sealing effect of the flow channel. The effect after encapsulation is as Figure 11 shown. The pin interface 442 of the biosensor can extend out horizontally for external wiring.

[0103] The biosensor 44 is installed at the bottom of the culture hole 423 of the first culture unit 421, which can real-time monitor the development-related parameters of the organoids 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 423 of the second culture unit 422 are transparent up and down and have observation windows, which can be used for optical observation and Raman spectroscopy measurement.

[0104] The culture chip 4 is placed in the culture chamber 1 and positioned by the limit block 37 to ensure that the culture holes 423 are within the field of view of the observation window of the culture chamber 1. When the upper cover 20 of the culture chamber 1 is closed, the flexible PCB of the biosensor of the culture chip 4 can just be located between the annular sealing ring 25 and the plane of the base 30, without affecting the airtightness of the entire chamber and ensuring the internal gas environment.

[0105] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and 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 their equivalents, the present invention is not limited to specific details.

Claims

1. An organoid culture device, characterized in that, include: A culture chamber, comprising a base assembly and an upper cover assembly rotatably connected to the base assembly, wherein the upper cover assembly comprises an upper cover and an upper heating film, an upper window, a temperature and humidity sensor and a locking mechanism arranged on the upper cover, and the base assembly comprises a base and a lower heating film, a temperature sensor, a humidifying dish and a lower window arranged on the base; the locking mechanism is used to cooperate with the base to realize the locking of the upper cover on the base; and a culture chip, which is arranged inside the culture chamber and between the upper window and the lower window; The culture chip comprises 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, the culture unit includes N culture holes arranged in a line and passing through the layer sheet body from top to bottom, 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 connected to the liquid inlet main channel, N distribution channels formed on the liquid inlet branch channel and connected to the inner side of the N culture holes one by one, N collection channels connected to the outer side of the N culture holes one by one, and a liquid discharge channel connected to 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; The biosensor is in the shape of a thin sheet, and has N detection plates at the end thereof that match the number and position of culture wells in the first culture unit of the two culture units. The N detection plates extend one by one under the N culture wells in the first culture unit to monitor the biological parameters of the organoids in the N culture wells in the culture unit and provide reverse electrical stimulation.

2. The organoid culture device according to claim 1, wherein The surface and bottom of the upper window are provided with grooves, each of which has a glass sheet disposed therein, and there is a gap between the two glass sheets in the upper window to form an air layer; The surface and the bottom surface of the lower window are also provided with grooves, each of which is provided with a glass sheet, and there is a gap between the two glass sheets in the lower window to form an air layer.

3. The organoid culture device according to claim 2, wherein, The locking mechanism is arranged at the end of the upper cover, and the locking mechanism comprises a swing block rotatably connected to the end of the upper cover through a pin shaft, a paddle connected to the outer end of the swing block, a lock hook connected to the lower part of the swing block, a tension spring connected between the end of the upper cover and the swing block, a limit handle connected to the end of the upper cover and located above the paddle, and a positioning pin connected to the bottom of the upper cover, and a positioning hole for the positioning pin to be inserted into is provided on the base; A barb for cooperating with the lock hook is provided at a corresponding position on the base below the lock hook; A waist hole is provided at the lower part of the swing block, and the locking hook is connected to the waist hole by screws, so that the height of the locking hook in the vertical direction can be adjusted; An annular sealing ring is arranged on the bottom edge of the upper cover.

4. The organoid culture device according to claim 3, wherein, Two limiting blocks are arranged along the diagonal direction on the periphery of the lower window to limit the position of the culture chip.

5. The organoid culture device according to claim 4, wherein, A gas interface pipe is provided on the side of the humidifying dish, and the gas outlet end of the gas interface pipe goes deep into the bottom of the humidifying dish, so that the gas discharged from the gas interface pipe is humidified by the water in the humidifying dish and then input into the culture chip; A plurality of liquid interface tubes are also arranged between the humidifying dish and the lower window to realize the perfusion of the culture liquid in the culture chip.

6. The organoid culture device according to claim 1, characterized in that, The upper glass sheet is provided with a liquid inlet hole located in the middle, two liquid outlet holes located on both sides of the liquid inlet hole, a plurality of screw holes and a first air hole; A liquid inlet pipe with its bottom extending into the liquid inlet main channel is inserted into the liquid inlet hole, and a liquid discharge pipe with its bottom extending into the liquid discharge channel is inserted into the liquid outlet hole. The liquid inlet pipe and the liquid discharge pipe are respectively connected to corresponding liquid path interface pipes.

7. The organoid culture device according to claim 6, characterized in that, The bottom surface of the sheet body below the first culture unit bulges downward to form a step surface, and the bottom surface of the step surface is provided with N rectangular grooves corresponding to the N culture holes in the first culture unit and used to accommodate the detection sheet of the biosensor.

8. The organoid culture device according to claim 7, wherein The thickness of the lower glass sheet is equal to the height of the protrusion of the step surface, and the lower glass sheet is arranged below the second culture unit of the two culture units so as to be flush with the step surface and can seal the bottoms of all culture wells in the second culture unit; The lower glass sheet is provided with a plurality of second air holes.

9. The organoid culture device according to claim 8, characterized in that, 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.

10. The organoid culture device according to claim 9, characterized in that, The area directly below the first culture unit on the packaging base 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 is also provided with a plurality of third air holes.

11. The organoid culture device according to claim 10, wherein, The second air hole corresponds to the third air hole on the packaging bottom plate in upper and lower positions, and the first air hole, the second air hole and the third air hole are all arranged away from the culture hole.

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